Optimizing Polyacrylamide Gel Electrophoresis for Accurate Protein Separation

Protein gel electroporesis handbook Techniques and tools for publication-quality results Strategies and solutions to drive your success Protein gel electrophoresis is a simple way to separate proteins prior to downstream detection or analysis, and is a critical step in most workflows that isolate, identify, and characterize proteins. In this handbook, you will find information on selecting the appropriate separation scheme, choosing the right gel and equipment, and preparing samples for analysis. Also included are troubleshooting guides, how-to videos, selection guides, and buffer recipes to help you achieve optimal results. Our portfolio of highquality protein electrophoresis products unites gels, stains, molecular weight markers, running buffers, and blotting products to provide you with a range of options designed for your experiments. See a complete listing of all available products and more at thermofisher.com/separate Contents Electrophoresis overview 4 Choosing the right protein gel 8 Bis-Tris chemistry vs. Tris-glycine chemistry 8 Gel selection guide 11 Precast protein gels 13 Handcast gel system 30 Preparing samples and selecting buffers 32 Protein extraction and clean-up methods 33 Protein quantitation 36 Electrophoresis buffers and reagents 38 Buffer recipes 39 Estimating protein sizes 42 Protein ladders and standards 43 Choosing the electrophoresis chamber and power supply 48 Electrophoresis chamber system selection guide 49 Electrophoresis chambers 50 Power supplies 58 Running gels 60 Gel running conditions 60 Staining gels 61 Protein stains 62 Protein stain selection guides 66, 68 Gel imaging and documentation 70 Western blotting 72 Transfer and detection 72 Appendix 73 Protocol quick references 73 Troubleshooting tips 74 Ordering information 78 Electrophoresis overview Electrophoresis is defined as the transport of charged molecules through a solvent by an electric field. Electrophoresis is a simple, rapid, and sensitive analytical tool for separating proteins and nucleic acids. Any charged ion or molecule will migrate when placed in an electric field. Most biological molecules carry a net charge at any pH other than at their isoelectric point and will migrate at a rate proportional to their charge density. The mobility of a biological molecule through an electric field will depend on the following factors: • Field strength • Net charge on the molecule • Size and shape of the molecule • Ionic strength • Properties of the matrix through which the molecules migrate (e.g., viscosity, pore size) 4 Support matrix Two types of support matrices are commonly used in electrophoresis: polyacrylamide and agarose. The support matrices act as porous media and behave like a molecular sieve. Separation of molecules is dependent upon the gel pore size of the support matrix used. Agarose has a large pore size and is ideal for separating macromolecules such as nucleic acids and protein complexes. Polyacrylamide has a smaller pore size and is ideal for separating most proteins, peptides, and smaller nucleic acids. Polyacrylamide gel electrophoresis (PAGE) Polyacrylamide gels are generated by the polymerization of acrylamide monomers. These monomers are crosslinked into long chains by the addition of bifunctional compounds such as N,N´-methylenebisacrylamide (bis), which react with the free functional groups of the chain termini. The concentration of acrylamide and bisacrylamide determines the pore size of the gel. The higher the acrylamide concentration, the smaller the pore size, resulting in resolution of lower molecular weight molecules and vice versa. PAGE allows one to separate proteins for different applications based on: • The acrylamide matrix • Buffer systems • Electrophoresis conditions ? Did you know Arne Tiselius won the Nobel Prize in Chemistry for electrophoretic analysis of serum proteins in 1948 The acrylamide matrix Linear vs. gradient gels Gels that have a single acrylamide percentage are referred to as linear gels, and those with a range are referred to as gradient gels. The advantage of using a gradient gel is that it allows the separation of a broader range of proteins than does a linear gel. Continuous vs. discontinuous gels Researchers occasionally refer to gels as continuous or discontinuous. A continuous gel is a gel that has been formed from a single acrylamide solution in the entire gel cassette. A discontinuous gel is formed from two acrylamide solutions: a small, low-percentage stacking gel where the protein wells reside, and a larger portion of gel that separates the proteins. In the traditional Tris-glycine protein gel system, the proteins are stacked in the stacking gel between the highly mobile leading chloride ions (in the gel buffer) and the slower, trailing glycine ions (in the running buffer). The reason for using the stacking gel is to improve the resolution of the bands in the gel. These stacked protein bands undergo sieving once they reach the separating gel. Mini vs. midi protein gels Commercial gels are available in two size formats: mini gels and midi gels. Both gels have similar run lengths, but midi gels are wider than mini gels, allowing midi gels to have more wells or larger wells. The additional wells in the midi gels permit more samples or large sample volumes to be loaded onto one gel. Buffer systems Electrophoresis is performed using continuous or discontinuous buffer systems. A continuous buffer system utilizes only one buffer in the gel and running buffer. A discontinuous buffer system utilizes a different gel buffer and running buffer [1]. This system may also use two gel layers of different pore sizes and different buffer composition (the stacking and separating gel). Electrophoresis using a discontinuous buffer system results in concentration of the sample in the stacking gel and higher resolution as a result. Electrophoresis conditions The separation of proteins is dependent on the electrophoresis conditions used, some of which are described below. Denaturing conditions (SDS-PAGE) Electrophoresis is performed under denaturing conditions using an anionic detergent such as sodium dodecyl sulfate (SDS). SDS denatures and unfolds the protein by wrapping around the hydrophobic portions. SDS binds at a ratio of ~1.4 g SDS per gram of protein. The resultant SDS–protein complexes are highly negatively charged and are resolved in the gel based on their size. Nondenaturing conditions (native PAGE) Electrophoresis is performed under nondenaturing (native) conditions using buffer systems that maintain the native protein conformation, subunit interaction, and biological activity. During native electrophoresis, proteins are separated based on their charge-to-mass ratios. Reducing conditions Electrophoresis is performed under reducing conditions using reducing agents such as dithiothreitol (DTT), β-mercaptoethanol (β-ME), or tris(2-carboxyethyl)phosphine (TCEP). The reducing agents completely unfold the denatured proteins into their subunits by cleaving the disulfide bonds between cysteine residues. 5 1D vs. 2D PAGE The most common form of protein gel electrophoresis is comparative analysis of multiple samples by one-dimensional (1D) electrophoresis, in which samples are loaded into wells, a current is applied to separate the proteins, and the resulting migration of the protein bands is visualized by staining or on a western blot. Multiple components of a single sample can be resolved most completely by two-dimensional electrophoresis (2D-PAGE). The first dimension separates proteins according to their native isoelectric point (pI) using a form of electrophoresis called isoelectric focusing (IEF). The second dimension separates proteins by mass using ordinary SDS-PAGE. 2D PAGE provides the highest resolution for protein analysis and is an important technique in proteomic research, where resolution of thousands of proteins on a single gel is sometimes necessary. The main focus of this handbook is on 1D electrophoresis. Protein gel chemistries PAGE utilizes a discontinuous buffer system to concentrate or “stack” samples into a very sharp zone in the stacking gel at the beginning of the run. In a discontinuous buffer system, the primary anion in the gel is different (or discontinuous) from the primary anion in the running buffer. Invitrogen™ Bolt™ Bis-Tris Plus gels, Invitrogen™ NuPAGE™ Bis-Tris and Tris-acetate gels, and the Laemmli system–based Invitrogen™ Novex™ Tris-Glycine gels are examples of discontinuous buffer systems and work in a similar fashion. However, Bis-Tris and Tris-acetate systems operate at a lower pH as a result of the ions that are in the system. Glycine (trailing ion) Protein/SDS complex (stacked proteins) Chloride (leading ion) Progression of run Common ion is Tris, present in the gel and running bu ers Figure 1. Tris-glycine gel system. • Gel buffer ions are Tris and chloride (pH 8.7) • Running buffer ions are Tris, glycine, and SDS (pH 8.3) • Gel operating pH is 9.5 Tris-glycine chemistry In a Tris-glycine system (Figure 1), three ions are primarily involved: • Chloride (–), supplied by the gel buffer, serves as the leading ion because it has the highest attraction to the anode relative to other anions in the system. • Glycine (–), the primary anion provided by the running buffer, serves as the trailing ion, because it is only partially negatively charged and remains behind the more highly charged chloride ions in a charged environment. • Tris base (+), a common ion present in both the gel and the running buffers. During electrophoresis, the gel and buffer ions in the Tris-glycine system form an operating pH of 9.5 in the separating region of the gel. 6 Bis-Tris chemistry In the case of the Bis-Tris system (Figure 2), three ions are primarily involved: • Chloride (–) supplied by the gel buffer, serves as the fastmoving leading ion. • MES or MOPS (–) (depending on the running buffer choice) serves as the trailing ion. – MES: 2-(N-morpholino) ethane sulfonic acid – MOPS: 3-(N-morpholino) propane sulfonic acid • Bis-Tris (+) acts as the common ion present in the gel while Tris (+) is provided by the running buffer. The combination of a lower-pH gel buffer (pH 6.4) and running buffer (pH 7.3–7.7) leads to a significantly lower operating pH (pH 7.0) during electrophoresis, resulting in better sample integrity and gel stability. Tris-acetate chemistry With the Tris-acetate system (Figure 3), three ions are primarily involved: • Acetate (–), the leading ion from the gel buffer • Tricine (–), the trailing ion from the running buffer • Tris (+), the common ion (in both gel and running buffer) This system also operates at a significantly lower pH than the Tris-glycine system, resulting in fewer gel-induced protein modifications. MES or MOPS (trailing ion) Protein/SDS complex (stacked proteins) Progression of run Common ion is Bis-Tris, present in the gel Chloride (leading ion) Tricine (trailing ion) Protein/SDS complex (stacked proteins) Acetate (leading ion) Progression of run Common ion is Tris, present in the gel and running buer Figure 2. Bis-Tris gel system. • Gel buffer ions are Bis-Tris and chloride (pH 6.4) • Running buffer ions are Tris, MES or MOPS, and SDS (pH 7.3) • Gel operating pH is 7.0 Figure 3. Tris-acetate gel system. • Gel buffer ions are Tris and acetate (pH 7.0) • Running buffer ions are Tris, tricine, and SDS (pH 8.3) • Gel operating pH is 8.1 Tricine chemistry The tricine system (not pictured) is a modification of the traditional Tris-glycine gel system that uses a discontinuous buffer system specifically designed for the resolution of low molecular weight proteins in the range of 2−20 kDa. As a result of reformulating the Laemmli running buffer and using tricine in place of glycine, SDS-polypeptides form behind the leading ion front rather than running with the SDS front, thus allowing for their separation into discrete bands. 7 The most widely used gel system for separating a broad range of proteins by SDS-PAGE is Tris-glycine gels (Laemmli system), comprising a stacking gel component that helps focus the proteins into sharp bands at the beginning of the electrophoretic run and the resolving gel component that separates the proteins based on size. This classic system uses a discontinuous buffer system where the pH and ionic strength of the buffer used for running the gel (Tris, pH 8.3) is different from the buffers used in the stacking gel (Tris, pH 6.8) and resolving gel (Tris, pH 8.8). The highly alkaline operating pH of the Laemmli system may cause band distortion, loss of resolution, or artifact bands (Figure 4). The major causes of poor band resolution with the Laemmli system are: • Hydrolysis of polyacrylamide at the high pH of the resolving gel, resulting in a short shelf life of 8 weeks • Chemical alterations such as deamination and alkylation of proteins due to the high pH of the resolving gel • Reoxidation of reduced disulfides from cysteine-containing proteins, as the redox state of the gel is not constant • Cleavage of Asp-Pro bonds of proteins when heated at 100°C in Laemmli sample buffer, pH 5.2 Unlike traditional Tris-glycine gels, Invitrogen™ NuPAGE™ and Bolt™ gels are Bis-Tris HCI–buffered (pH 6.4) and have an operating pH of about 7.0. The neutral operating pH of the Bis-Tris system provides the following advantages over the Laemmli system: • Longer shelf life of 16 months, due to improved gel stability • Improved protein stability during electrophoresis at neutral pH, enabling sharper band resolution and accurate results [2] • Complete reduction of disulfides under mild heating conditions (70°C for 10 minutes) and absence of cleavage of Asp-Pro bonds • Reduced state of the proteins maintained during electrophoresis and blotting of the proteins when using Invitrogen™ NuPAGE™ Antioxidant or Invitrogen™ Bolt™ Antioxidant Denaturing gel systems NuPAGE Bis-Tris and Bolt Bis-Tris are well suited for separating a broad range of protein sizes. To separate high-abundance proteins, select our robust Invitrogen™ Novex™ Tris-Glycine gel chemistry. Tris-acetate gel chemistry, offered in Invitrogen™ NuPAGE™ Tris-Acetate gels, is recommended for the separation of high molecular weight proteins up to 500 kDa. Tricine gel chemistry is designed for the separation of low molecular weight proteins and peptides. Invitrogen™ Novex™ tricine gels enable increased resolution of proteins with molecular weights as low as 2.5 kDa. Figure 4. Protein separation using (A) an Invitrogen™ Bolt™ Bis-Tris Plus gel and (B) Bio-Rad’s Tris-glycine gel. 1 2 A 1 2 33 44 55 66 77 88 99 1100 B 11 22 33 44 55 66 77 88 99 1100 Choosing the right protein gel Bis-Tris chemistry vs. Tris-glycine chemistry 8 Other gel systems Native gels In native polyacrylamide gel electrophoresis, proteins are separated according to the net charge, size, and shape of their native structure. Electrophoretic migration occurs because most proteins carry a net negative charge in alkaline running buffers, with proteins of greater negative charge density migrating faster. At the same time, the sieving effect of the gel matrix regulates the migration of proteins according to their size and threedimensional shape. The Invitrogen™ NativePAGE™ Bis-Tris Gel System is based on the blue native polyacrylamide gel electrophoresis (BN PAGE) technique developed by Schägger and von Jagow, which overcomes the limitations of traditional native electrophoresis by providing a near-neutral operating pH and detergent compatibility. In this specific system, the Coomassie G-250 dye binds to proteins and confers a net negative charge while maintaining the proteins in their native state. NativePAGE gels are designed to separate proteins up to 10,000 kDa. Because no denaturants are used in the NativePAGE system, protein subunits are generally retained and information can be gained about the quaternary structure. In addition, some proteins retain their enzymatic activity following separation using the NativePAGE system. Tris-glycine and Tris-acetate gel systems can also be used for native PAGE when used in the absence of SDS in sample and running buffers. IEF gels Isoelectric focusing (IEF) is a technique designed to separate proteins according to their isoelectric point (pI) rather than molecular weight. The pI is the pH at which a protein has no net charge and no longer moves in an electric field. These gels can be used to determine the pI or to detect minor changes in a protein due to deamination, phosphorylation, or glycosylation. They can also resolve different proteins of similar size that cannot be resolved on standard SDS-PAGE gels. Zymogram gels Invitrogen™ Zymogram gels are composed of gelatin and are used to characterize proteases that utilize gelatin as a substrate, such as matrix metalloproteases, lipases, and other proteases. Samples are run under denaturing conditions, but due to the absence of reducing agents, proteins can undergo renaturation under appropriate buffer conditions (e.g., Invitrogen™ Novex™ Zymogram Renaturing Buffer). Proteolytic proteins present in the sample consume the substrate in the presence of added divalent metal cations (e.g., Invitrogen™ Novex™ Zymogram Developing Buffer). The gels are then stained to generate clear bands where the substrate has been digested, against a background stained blue. High-throughput gel electrophoresis High-throughput gel electrophoresis expands the number of protein samples that can be analyzed in a given time and is especially useful for screening recombination products and protein profiling. While midi gels enable higher-throughput electrophoresis and western blotting than mini gels, the Invitrogen™ E-PAGE™ High- Throughput (HTP) Precast Gel System is specially designed for fast, bufferless HTP protein analysis. Invitrogen™ E-PAGE™ gels are self-contained, precast gels that include a gel matrix and electrodes packaged inside a disposable cassette. E-PAGE gels are available in 48-well or 96-well formats. These gels are run using the Invitrogen™ E-Gel™ Power Snap Plus Electrophoresis System, which provides both electrophoresis and real-time gel imaging functions in a single benchtop instrument. 9 Precast vs. handcast gels Traditionally, researchers poured their own gels using standard recipes that are widely available in the protein methods literature. Today, more researchers rely on the convenience and consistency of commercially available, ready-to-use precast gels. We offer precast gels in a variety of percentages, including difficult-to-pour gradient gels that provide excellent resolution and separate proteins over the widest possible range of molecular weights. Precast gels are also available with several different buffer formulations (e.g., Tris-glycine, Tris-acetate, Bis-Tris, and tricine), which are designed to optimize shelf life, run time, and protein resolution. More importantly, precast polyacrylamide gels minimize the need to work with acrylamide, which is a known neurotoxin and suspected carcinogen. However, some scientists need unique gel formulations not available in precast gel formats. In those cases, the gel-pouring process can be made easier by using newer, leak-free handcast systems, such as the Invitrogen™ SureCast™ system, or by using preassembled empty gel cassettes. Choosing the right gel percentage In general, the size of the molecule being separated should dictate the acrylamide or agarose percentage you choose. Use a lower percentage gel to resolve larger molecules and a higher percentage gel to resolve smaller ones. The exception to this rule is when performing isoelectric focusing. Refer to the gel migration charts throughout this chapter to find the gel best suited for your application. As a general rule, molecules should migrate through about 70% of the length of the gel for the best resolution. When protein molecular weights are wide ranging or unknown, gradient gels are usually the best choice. Choosing a well format and gel thickness Two thicknesses (1.0 mm and 1.5 mm) are available for popular gel types. If loading large sample volumes (>30 μL), a thicker gel (1.5 mm) may be more appropriate. Another consideration for large sample volumes is choosing a wedge-well format that is found in many of our protein gels. When blotting, remember that proteins will transfer more easily from a 1.0 mm thick gel than from a 1.5 mm thick gel. We offer most of our polyacrylamide gels in twelve different well formats (mini gels: 17-well, 15-well, 12-well, 10-well, 9-well, 1-well, 2D/preparative well, IPG well; midi gels: 12+2-well, 20-well, 26-well). WedgeWell format wells Invitrogen™ WedgeWell™ format wells are wedge-shaped wells that double the sample loading capacity compared to traditional wells. WedgeWell format gels are available in mini and midi sizes and in Bis-Tris, Tris-glycine, and Tris-acetate chemistries, offering: • Easy sample loading—larger well openings make loading a breeze even with standard pipette tips, and reduce sample spillover and cross-well contamination • Increased sample load—easier detection of dilute samples and low-abundance proteins • Faster run times—proteins can be separated up to 10 minutes faster than traditional gels To learn more about WedgeWell format wells, watch the video at thermofisher.com/proteingels WedgeWell format WedgeW ewlle flol rmat well ConventioCnoanl vweenlltional well 10 ? Did you know Over 55 years ago, Ulrich K. Laemmli first published on SDS-PAGE as a method for cleavage analysis of structural proteins in bacteriophage T4. Gel selection guide Getting started with SDS-PAGE Use the decision tree below to guide your choice of gel based on the protein molecular weight and the gel format. Still unsure of what gel to choose? Use our interactive product selector at thermofisher.com/proteingelguide. If you would like to replace your current precast gels from another supplier with Invitrogen™ gels, go to thermofisher.com/proteingelconversion. Specialized protein gels Description Type of separation Use for NativePAGE gels Net charge, size, and shape of native protein Analysis of native proteins or protein complexes Novex IEF gels Isoelectric point of native protein Determination of isoelectric point Zymogram gels SDS-PAGE Characterization of proteases that utilize casein or gelatin as a substrate E-PAGE high-throughput gel system SDS-PAGE Fast, high-throughput protein separation Gel selection guide What is the approximate molecular weight of the target protein? Novex Tricine gels NuPAGE Tris-Acetate gels Bolt Bis-Tris Plus gels NuPAGE Bis-Tris gels 2.5–40 kDa Low molecular weight 6–250 kDa Broad-range molecular weight 40–500 kDa • High molecular weight • WedgeWell format • Faster run times • Longer shelf life • Less protein degradation • Lower list price • WedgeWell format • Tris-glycine gels preferred • Highest performance • Standard format (mini) • WedgeWell format (midi) • Highest performance • WedgeWell format • Fastest Bis-Tris gel on the market Mini and midi format gels Mini and midi format gels Mini and midi format gels Novex Tris-Glycine gels 11 Protein gel performance guarantee We stand behind the quality of our high-performance protein gels. Purchase Invitrogen protein gels with confidence, knowing that our gels are backed by our protein gel performance guarantee. If an Invitrogen protein gel does not perform in your experiment as described on our website or Certificate of Analysis, we will replace the product at no cost to you, or we will provide you with a credit for future purchase. Learn about our protein gel performance guarantee at thermofisher.com/proteingelguarantee Protein gel welcome packs Protein gel welcome packs are cost-saving product combinations that contain all you need for outstanding protein separation. Protein gel welcome packs include: • Mini or midi gel tank • 2 boxes of mini or midi gels • Running buffer • Sample buffer • Reducing agent • Prestained ladder Options are available that include mini blot modules and WedgeWell format gels. Explore available welcome packs at thermofisher.com/proteingelwelcome 12 Bolt Bis-Tris Plus and NuPAGE Bis-Tris gels are precast polyacrylamide gels designed for optimal separation of a broad molecular weight range of proteins under denaturing conditions (Figures 5–7). These gels help deliver consistent performance with a neutral-pH environment that minimizes protein degradation, resulting in sharper, well-resolved bands. Additionally, preserving protein integrity becomes particularly important when separating low-abundance proteins. Bolt Bis-Tris Plus and NuPAGE Bis-Tris gels are a good choice when protein integrity is crucial. Bolt Bis-Tris Plus gels are available in the mini gel format and NuPAGE Bis-Tris gels are available in mini and midi gel formats, as well as multiple thicknesses. View the video to see how easy it is to run an Invitrogen gel here. Review the quick reference protocol for Bolt gels here. Review the quick reference protocol for NuPAGE Bis-Tris mini gels here. Learn more at thermofisher.com/nupage Bolt Bis-Tris Plus and NuPAGE Bis-Tris gels Neutral-pH gel systems for optimal separation Bolt Bis-Tris Plus and NuPAGE Bis-Tris gels offer: • Excellent protein integrity—neutral-pH formulation minimizes protein modifications (Figure 6) • Fast run times—separate proteins in 20–35 minutes • High sample volume capacity—WedgeWell format wells double the sample loading capacity and make it easy to load samples • More efficient western blot transfer—neutral pH prevents reoxidation of reduced samples during protein transfer • Long shelf life—up to 16 months at room temperature NuPAGE Bis-Tris gels have been referenced in >90,000 publications. Figure 5. Excellent protein loading capacity of NuPAGE Bis-Tris Midi gels, WedgeWell format. Decreasing loads of HEK293 cell lysate in the NuPAGE Bis-Tris Midi gel show excellent separation, even at high protein loads. Streaking and retention of protein in the wells can be observed in the Bio-Rad gel when protein loads exceed 30 μg. EPHB3 GST HCK FLT1 IKK MAPK14 DDR2 MagicMark standard EPHB3 GST HCK FLT1 IKK MAPK14 DDR2 MagicMark standard Bolt Bis-Tris Plus gel Bio-Rad TGX gel Figure 6. Bolt Bis-Tris Plus mini gels help provide better western blotting results. A western blot of a Bolt gel shows clean, sharp protein bands corresponding to only full-length proteins, whereas a western blot of a Bio‑Rad™ TGX™ gel shows multiple low molecular weight degradation products. NuPAGE Bis-Tris 4–12% midi gel, WedgeWell format Lysate (μg): 60 50 40 30 20 10 5 2 1 0.5 Bio-Rad 4–20% Criterion TGX midi gel Lysate (μg): 60 50 40 30 20 10 5 2 1 0.5 Bio-Rad 4–20% Criterion TGX midi gel Lysate (μg): 60 50 40 30 20 10 5 2 1 0.5 Bio-Rad 4–20% Criterion TGX midi gel Lysate (μg): 60 50 40 30 20 10 5 2 1 0.5 13 Figure 7. Improved detection of low-abundance proteins. Serial dilutions of a HEK293 cell lysate starting at 250 ng/μL were loaded in standard and WedgeWell format NuPAGE Bis-Tris Midi Protein Gels. After transfer and blot processing, fluorescent detection was performed with the Invitrogen™ iBright™ FL1500 Imaging System. 250 ng/μL 166.7 ng/μL 111.1 ng/μL 74.1 ng/μL 49.4 ng/μL 32.9 ng/μL 21.9 ng/μL 14.6 ng/μL 9.8 ng/μL 6.5 ng/μL 4.3 ng/μL 2.9 ng/μL 1.9 ng/μL Standard format (25 μL sample) WedgeWell format (60 μL sample) 250 ng/μL 166.7 ng/μL 111.1 ng/μL 74.1 ng/μL 49.4 ng/μL 32.9 ng/μL 21.9 ng/μL 14.6 ng/μL 9.8 ng/μL 6.5 ng/μL 4.3 ng/μL 2.9 ng/μL 1.9 ng/μL Standard format (25 μL sample) WedgeWell format (60 μL sample) Calreticulin Vinculin 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Normalized signal Normalized signal Lysate concentration (μg/μL) Calreticulin Lysate concentration (μg/μL) 0.250 0.167 0.111 0.074 0.049 0.033 0.022 0.015 0.010 0.007 0.250 0.167 0.111 0.074 0.049 0.033 0.022 0.015 0.010 Standard format WedgeWell format Standard format WWeeddggeeWWeellll ffoorrmmaatt 14 Bolt Bis-Tris Plus mini gel specifications • Shelf life: 16 months • Run time: 20–30 minutes on average; 15 minutes with rapid protocols • Separation range: 0.3–260 kDa • Polyacrylamide concentrations: fixed 8%, 10%, and 12%; gradient 4–12% • Gel dimensions: 8 x 8 cm (1 mm thick) • Maximum sample volume per 10-well gel: ~60 μL, or two-thirds of the sample well volume Bolt Bis-Tris Plus gels Figure 8. Bolt Bis-Tris Plus gel migration chart. Optimal separation range is shown within the gray areas. The new Bolt™ system is wonderful. I am still amazed that I can run a PAGE gel in 23 minutes. The entire system is incredibly user-friendly, from the Bolt™ precast gels with wedged wells for ease of loading to the Mini Gel Tank system. The bands produced from the westerns were sharp and straight. I would and have highly recommended this system to anyone doing protein work. —Crystal M., large Canadian university For one of our projects in the lab, we resolve proteins by electrophoresis to determine the accumulation of ubiquitinated proteins following treatment with a proteasome inhibitor. When we resolved the ubiquitinated proteins using the Tris-glycine gels, we observed a smear. However, when we switched to resolving the ubiquitinated proteins using the Bolt Bis-Tris gels, we were delightfully surprised to observe individual protein bands in place of the smear. —Susan S., large U.S. university “ “ 15 NuPAGE Bis-Tris gels (denaturing separation) Figure 9. Migration patterns in NuPAGE Bis-Tris gels using Invitrogen™ Novex™ Sharp Prestained Protein Standard or Novex™ Sharp™ Unstained Protein Standard. The optimal separation range is shown within the gray areas. Review the quick reference guide for NuPAGE Bis-Tris Mini Gels here. * Shelf life varies depending on gel format. NuPAGE Bis-Tris gel specifications • Shelf life: up to 16 months* at room temperature (4–25°C) • Average run time: 25 minutes (MES buffer) • Separation range: 1.5–300 kDa • Polyacrylamide concentrations: fixed 8%, 10%, and 12%; gradient 4–12% • Gel dimensions: – Mini: 8 x 8 cm (1 or 1.5 mm thick) – Midi: 8 x 13 cm (1 mm thick) – Midi, WedgeWell format: 8 x 13 cm (1 mm thick) • Maximum sample volume per 10-well mini gel: 25 μL (1 mm thick); 37 μL (1.5 mm thick; up to 100 μL for WedgeWell format midi gels) Recommended products Thermo Scientific™ PageRuler™ Plus and Spectra™ prestained protein ladders are recommended for use with NuPAGE Bis-Tris gels for easy molecular weight determination. Visualize with Coomassie stain, silver stain, or fluorescent protein stains after electrophoresis (see “Staining gels” on page 60). 16 ? Did you know Timothy Updyke and Sheldon Engelhorn filed a patent for the neutral-pH Bis-Tris gel system in 1996. Figure 10. Better protein resolution and band sharpness with Novex Tris-Glycine Mini Gels, WedgeWell format. Protein ladder, purified proteins, and E. coli lysate were loaded on a gradient Novex 4−20% Tris-Glycine Mini Gel, WedgeWell format, and a Bio-Rad TGX 4–20% gradient gel. The Bio-Rad TGX gel displays numerous low molecular weight protein degradation products below major bands in lanes 3, 4, 7, and 8. These are not seen in the Novex Tris-Glycine gel. The Novex gel also displays better protein band sharpness and resolution of lysate than the Bio-Rad gel. Lanes 1, 10: 5 μL Mark12 Unstained Standard; lane 2: 10 μg E. coli lysate; lane 3: 6 μg catalase; lane 4: 6 μg carbonic anhydrase; lane 5: 6 μg lysozyme; lane 6: 6 μg hIgM; lane 7: 6 μg BSA; lane 8: 6 μg β-galactosidase; lane 9: 20 μg E. coli lysate. The sample volume for lanes 2–9 was 10 μL. Review the quick reference protocol for the Novex Tris-Glycine mini gels, WedgeWell format, here. Learn more at thermofisher.com/novexwedge Novex Tris-Glycine gels Figure 11. The protein loading capacity of Novex Tris-Glycine Plus midi gels, WedgeWell format, exceeds that ofBio-Rad™ Criterion™ TGX™ gels. The Bio-Rad gel shows streaking, smearing, and retention of protein in the well for protein loads above 20 μg of lysate. Highlights: • Wedge-shaped wells—easily load up to 100 μL of sample without sacrificing gel width or length (mini gel only) • High performance—excellent protein band resolution and sharpness • Improved shelf life—store gels for up to 12 months at 4°C • Fast run conditions—up to 10 minutes faster than traditional Tris-glycine gels • Flexible—compatible with native and denatured protein samples • Cost efficiency—load more samples in larger-format midi gels and save Load up to 100 μL of sample The Invitrogen™ Novex™ Tris-Glycine mini gels, WedgeWell™ format, and Invitrogen™ Novex™ Tris-Glycine Plus midi gels are polyacrylamide gels based on traditional Laemmli chemistry that enable the use of Laemmli sample and running buffers. Novex Tris-Glycine gels provide high-quality performance and separation of a wide range of proteins into well-resolved bands (Figures 10 and 11). Novex 4–20% Lysate (μg): Novex 4–20% Tris-Glycine Plus midi gel, WedgeWell format Bio-Rad 4–20% Criterion TGX midi gel Lysate (μg): 60 50 40 30 20 10 5 2 1 0.5 Lysate (μg): 60 50 40 30 20 10 5 2 1 0.5 Novex 4–20% Tris-Glycine Mini Gel, WedgeWell format Bio-Rad TGX 4–20% Gel 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Novex 4–20% Tris-Glycine Mini Gel, WedgeWell format Bio-Rad TGX 4–20% Gel 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 60 50 40 30 20 10 5 2 1 0.5 μg 60 50 40 30 20 10 5 2 1 0.5 μg 17 Recommended products For sample clean-up prior to electrophoresis, we recommend using the Thermo Scientific™ Pierce™ SDS-PAGE Sample Prep Kit. Buffers for denatured proteins: Invitrogen™ Novex™ Tris-Glycine SDS Sample Buffer and Tris-Glycine SDS Running Buffer, or Rapid Tris-Glycine SDS Running Buffer (Powder). Buffers for native proteins: Invitrogen™ Novex™ Tris-Glycine Native Sample Buffer and Tris-Glycine Native Running Buffer. PageRuler Plus and Spectra protein ladders are recommended for molecular weight determination with Novex Tris-Glycine gels. Novex Tris-Glycine gel specifications • Shelf life: up to 12 months at 4°C • Average run time: 45 minutes • Separation range: 8 kDa to 260 kDa • Polyacrylamide concentrations: fixed 6%, 8%, 10%, 12%, 14%, 16%; gradient 4–12%, 4–20%, 8–16%, 10–20% • Gel dimensions: – Mini, WedgeWell format: 8 x 8 cm (1 mm thick) – Midi, WedgeWell format: 8 x 13 cm (1 mm thick) • Maximum sample volume: – In 10-well mini gel, WedgeWell format: 60 μL – In 20-well midi gel with standard well format: 25 μL 18 Novex Tris-Glycine gels 66 kDa 10 20 30 40 50 60 70 80 90 100 6% 8% 10% 12% 14% 16% 200 kDa 116 kDa 97 kDa 97 kDa 66 kDa 66 kDa 55 kDa 36 kDa 36 kDa 36 kDa 36 kDa 31 kDa 31 kDa 21 kDa 21 kDa 21 kDa 14 kDa 14 kDa 6 kDa 6 kDa 6 kDa 6 kDa 116 kDa 97 kDa 200 kDa 200 kDa 200 kDa 200 kDa 200 kDa 116 kDa 116 kDa 116 kDa 116 kDa 116 kDa 66 k Da 97 kDa 55 kDa 55 kDa 97 kDa 66 kDa 55 kDa 31 kDa 21 kDa 14 kDa 36 kDa 97 kDa 66 kDa 55 kDa 31 kDa 4–12% 8–16% 4–20% 10–20% Large proteins (116 –500 kDa) Midsize proteins (20–250 kDa) Small proteins (3–60 kDa) Wide range (6–200 kDa) 200 kDa 97 kDa 36 kDa 31 kDa 21 kDa 66 kDa 55 kDa 200 kDa 200 kDa 200 kDa 116 kDa 116 kDa 116 kDa 97 kDa 97 kDa 66 kDa 66 kDa 66 kDa 55 kDa 55 kDa 55 kDa 36 kDa 36 kDa 36 kDa 31 kDa 31 kDa 31 kDa 21 kDa 21 kDa 14 kDa 14 kDa 6 kDa Percent length of gel Novex Tris-Glycine gels, WedgeWell format 97 kDa Figure 12. Migration patterns of a protein standard on Novex Tris-Glycine gels, WedgeWell format. Use this chart to select the proper gel for separating proteins based on size. Optimal resolution is achieved when protein bands migrate within the shaded regions. The standard represented here is the Mark12 Unstained Standard under denaturing conditions. 66 kDa 10 20 30 40 50 60 70 80 90 100 6% 8% 10% 12% 14% 16% 200 kDa 116 kDa 97 kDa 97 kDa 66 kDa 66 kDa 55 kDa 36 kDa 36 kDa 36 kDa 36 kDa 31 kDa 31 kDa 21 kDa 21 kDa 21 kDa 14 kDa 14 kDa 6 kDa 6 kDa 6 kDa 6 kDa 116 kDa 97 kDa 200 kDa 200 kDa 200 kDa 200 kDa 200 kDa 116 kDa 116 kDa 116 kDa 116 kDa 116 kDa 66 kD a 97 kDa 55 kDa 55 kDa 97 kDa 66 kDa 55 kDa 31 kDa 21 kDa 14 kDa 36 kDa 97 kDa 66 kDa 55 kDa 31 kDa 4–12% 8–16% 4–20% 10–20% Large proteins (116 –500 kDa) Midsize proteins (20–250 kDa) Small proteins (3–60 kDa) Wide range (6–200 kDa) 200 kDa 97 kDa 36 kDa 31 kDa 21 kDa 66 kDa 55 kDa 200 kDa 200 kDa 200 kDa 116 kDa 116 kDa 116 kDa 97 kDa 97 kDa 66 kDa 66 kDa 66 kDa 55 kDa 55 kDa 55 kDa 36 kDa 36 kDa 36 kDa 31 kDa 31 kDa 31 kDa 21 kDa 21 kDa 14 kDa 14 kDa 6 kDa Percent length of gel Novex Tris-Glycine gels, WedgeWell format 97 kDa 19 Recommended products Invitrogen™ HiMark™ Unstained and Prestained Protein Standards are specifically designed for large protein analysis on NuPAGE Tris-Acetate gels under denaturing conditions. Both standards offer a ready-to-load format and consist of 9 proteins with a size range of 40–500 kDa. Tris-acetate gel chemistry enables the optimal separation of high molecular weight proteins. NuPAGE Tris-Acetate gels offer a pH 8.1 environment that minimizes protein modifications and results in sharper bands. NuPAGE Tris-Acetate gels can also be run with Novex Tris-Glycine Native Running Buffer to resolve native proteins more effectively than a Tris-glycine gel system. NuPAGE Tris-Acetate gels and buffers are designed to allow: • Optimal separation of high molecular weight proteins (Figures 13 and 15) • Preservation of protein sample integrity using optimized sample preparation processes • More efficient western blot transfer—neutral pH prevents reoxidation of reduced samples during protein transfer (Figure 14) • Double the sample loading capacity with WedgeWell format wells Specifications • Shelf life: 8 months • Average run time: 35 minutes • Separation range: 30–400 kDa • Polyacrylamide concentrations: fixed 7%; gradient 3–8% • Gel dimensions: – Mini (traditional) and mini (WedgeWell format): 8 x 8 cm (1 or 1.5 mm thick) – Midi (traditional) and midi (WedgeWell format): 8 x 13 cm (1 mm thick) • Maximum sample volume per 10-well mini gel: 25 μL (1 mm thick); 37 μL (1.5 mm thick) Novex 4–20% Tris-Glycine mini gel, WedgeWell format NuPAGE 3–8% Tris-Acetate mini gel EGFR (~190 kDa) EGFR (~190 kDa) 20 μg 10 μg 5 μg 2.5 μg 1.25 μg 0.75 μg 0.62 μg 20 μg 6.7 μg 2.3 μg 0.74 μg 0.25 μg 83 ng 28 ng 9 ng Figure 14. Improved transfers of high molecular weight proteins enhance western detection sensitivity. Western blotting analysis of EGFR from A431 lysates transferred from a Novex 4–20% Tris-Glycine mini gel, WedgeWell format, and a NuPAGE 3–8% Tris-Acetate mini gel using the iBlot 2 Gel Transfer Device. NuPAGE Tris-Acetate gels High molecular weight protein separation Figure 13. NuPAGE Tris-Acetate gel electrophoresis. An Invitrogen™ NuPAGE™ Tris-Acetate 3–8% gel, WedgeWell format, was loaded as follows: lanes 1, 20: 5 μL Thermo Scientific™ PageRuler™ Unstained Broad Range Protein Ladder (Cat. No. 26630); lanes 2–7: 10 μg, 8 μg, 6 μg, 4 μg, 2 μg, and 1 μg HeLa cell lysate; lanes 8, 13: 5 μL Invitrogen™ Mark12™ Unstained Standard (Cat. No. LC5677); lanes 9–12: 240 ng, 180 ng, 120 ng, and 60 ng of protein mix containing β-galactosidase, lactate dehydrogenase, and lysozyme; lanes 14–19: 10 μg, 8 μg, 6 μg, 4 μg, 2 μg, and 1 μg E. coli lysate. Gel was stained with Invitrogen™ SimplyBlue™ SafeStain. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 250 kDa 150 100 70 50 40 30 5240 Figure 15. Migration patterns in NuPAGE Tris-Acetate gels. For optimal results, protein bands should migrate within the gray shaded areas. (A) Migration pattern of Invitrogen™ HiMark™ Unstained Protein Standard on a NuPAGE Tris-Acetate gel under denaturing conditions. (B) Migration pattern of Invitrogen™ NativeMark™ Unstained Protein Standard on a NuPAGE Tris-Acetate gel under native conditions. NuPAGE Tris-Acetate gels (denaturing separation) A NuPAGE Tris-Acetate gels (native separation) B Learn more about NuPAGE Tris-Acetate gels at thermofisher.com/trisacetate Review the quick reference protocol for NuPAGE Tris-acetate mini gels here. 21 Novex Tricine gels Recommended products Use Novex Tricine gels with the Thermo Scientific™ In-Gel Tryptic Digestion Kit for separation and digestion of peptides for mass spectrometry. Figure 17. Novex Tricine gel migration chart. Migration patterns of the Mark12 Unstained Standard are shown. High-resolution gels for peptide analysis and low molecular weight proteins The Invitrogen™ Novex™ tricine gel system is a modification of the Tris-glycine system in which tricine replaces glycine in the running buffer. This system uses a discontinuous buffer system specifically designed for the resolution of low molecular weight proteins (Figure 16). Advantages of Novex Tricine gels over Tris-glycine gels include: • Increased resolution of proteins with molecular weights as low as 2 kDa (Figure 17) • Improved compatibility with direct protein sequencing applications after transferring to PVDF membranes • Minimized protein modifications, due to the lower pH of the tricine buffering system Figure 16. Novex Tricine gel electrophoresis. Protein standards and samples were loaded at 10 μL sample volumes on Invitrogen™ Novex™ 10–20% Tricine Protein Gels. Electrophoresis was performed using the Mini Gel Tank at 200 V (constant). Sharp, straight bands were observed after staining with SimplyBlue SafeStain. Image was acquired using a flatbed scanner. Lane 1: SeeBlue Plus2 Prestained Standard; lane 2: 10 μg E. coli lysate; lane 3: Mark12 Unstained Standard (blend of 12 purified proteins); lane 4: 40 μg HeLa cell lysate; lane 5: 20 μg HeLa cell lysate; lane 6: 5 μg BSA; lane 7: 40 μg Jurkat cell lysate; lane 8: 5 μg GST fusion protein; lane 9: Novex Sharp Unstained Protein Standard; lane 10: 5 μg β-galactosidase. Review the protocol for Novex Tricine gels here. Learn more at thermofisher.com/tricine Novex Tricine gels 22 How Novex Tricine gels work In the traditional Tris-glycine protein gel system, the resolution of smaller proteins (<10 kDa) is hindered by the continuous accumulation of free dodecyl sulfate (DS) ions from the SDS contained in the sample and running buffers in the stacking gel, which causes mixing of the DS ions with smaller proteins and results in fuzzy bands and decreased resolution. The mixing also interferes with the fixing and staining of smaller proteins. The Novex Tricine gel system uses a low pH in the gel buffer and substitutes tricine for glycine in the running buffer. The smaller proteins and peptides that migrate with the stacked DS ions in the Tris-glycine gel system are well separated from DS ions in the Novex Tricine gel system, offering sharper bands and higher resolution. Good to know ? Did you know Sample preparation is not the only factor that can result in poorly resolved bands. You can minimize protein degradation by using gels with neutral-pH chemistry. NativePAGE gels Recommended products The NativeMark Unstained Protein Standard is recommended for use with native gel chemistries, including our Tris-glycine, Tris-acetate, and NativePAGE gel systems. This standard offers a wide molecular weight range of 20–1,200 kDa, and the 242 kDa β-phycoerythrin is visible as a red band after electrophoresis for reference (prior to staining). NativePAGE Bis-Tris gels Excellent resolution of native proteins and protein complexes The Invitrogen™ NativePAGE™ Bis-Tris gel system is based on the blue native polyacrylamide gel electrophoresis (BN PAGE) technique that uses Coomassie G-250 dye as a charge shift molecule that binds to proteins and confers a negative charge without denaturing the proteins (Figure 18). This technique overcomes the limitations of traditional native electrophoresis by providing a near-neutral operating pH and detergent compatibility. The near-neutral (pH 7.5) environment of the NativePAGE gel system during electrophoresis results in maximum protein and gel matrix stability, enabling better band resolution than other native gel systems. A gel migration chart is shown in Figure 19. The NativePAGE gel system is designed for: • A wide resolving range—from 15 kDa to >10 MDa, regardless of isoelectric point • Neutral-pH separation—the native state of protein complexes is better preserved • Excellent performance—higher resolution than Tris-glycine native electrophoresis Figure 18. NativePAGE gel electrophoresis. Two-fold serial dilutions of protein extracts were run on an Invitrogen™ NativePAGE™ 3–12% Bis-Tris Protein Gel using a Mini Gel Tank. Following electrophoresis, the gel was stained with Coomassie dye and imaged using a flatbed scanner. Lanes 1, 10: blank; lanes 2, 6: 5 μL NativeMark Unstained Protein Standard; lanes 3–5: 10, 5, and 2.5 μg spinach chloroplast extract; lanes 7–9: 10, 5, and 2.5 μg bovine mitochondrial extract. Learn more at thermofisher.com/nativepage Review the protocol for NativePAGE Bis-Tris gels here. Figure 19. NativePAGE gel migration chart. Migration patterns of the NativeMark Unstained Protein Standard on NativePAGE gels are shown. 23 ? Did you know The blue native polyacrylamide gel electrophoresis technique was developed by Hermann Schägger and Gebhard von Jagow in 1991. 24 Novex IEF gels Precast gels for isoelectric point determination Specifications • Shelf life: 6 months • Run time: 2.5 hours • Separation range: – pH 3–10 gels: pI performance range is 3.5–8.0 – pH 3–7 gels: pI performance range is 3.0–7.0 4.5 6.0 7.4 8.0 1 2 3 4 5 6 7 8 9 10 pl Figure 20. Novex IEF gel electrophoresis. A 2-fold dilution series of Invitrogen™ IEF Marker 3–10 was run in duplicate on an Invitrogen™ Novex™ pH 3–10 IEF Protein Gel using a Mini Gel Tank. The IEF Marker 3–10 consists of proteins with a variety of isoelectric points; these proteins include lectin (pI = 7.8, 8.0, and 8.3), myoglobin from horse muscle (pI = 6.9 and 7.4), carbonic anhydrase from bovine erythrocytes (pI = 6.0), β-lactoglobulin from bovine milk (pI = 5.2 and 5.3), soybean trypsin inhibitor (pI = 4.5), and glucose oxidase (pI = 4.2). After electrophoresis, the gel was fixed and stained using Coomassie R-250 dye. Gel imaging was performed with a flatbed scanner. Volumes of IEF Marker 3–10 loaded: Lanes 1, 6: 20 μL; lanes 2, 7: 10 μL; lanes 3, 8: 5 μL; lanes 4, 9: 2.5 μL; lanes 5, 10: blank. Learn more at thermofisher.com/ief Isoelectric focusing (IEF) is an electrophoresis technique that separates proteins based on their isoelectric point (pI). The pI is the pH at which a protein has no net charge and does not move in an electric field. Invitrogen™ Novex™ IEF gels effectively create a pH gradient so proteins separate according to their unique pI (Figures 20 and 21). These gels can be used for pI determination or for detection of minor changes in a protein due to deamination, phosphorylation, or glycosylation, and can resolve different proteins of similar size that cannot be resolved on standard SDS-PAGE gels. When used with our convenient, preoptimized buffers, solubilizers, and molecular weight markers, Novex IEF gels can provide: • Accurate pI determination • Clear, sharp bands for easy identification of protein modifications • Higher resolution of slight differences in size when used in combination with SDSPAGE for 2D electrophoresis • Polyacrylamide concentration: fixed 5% • Gel dimensions: 8 x 8 cm (1 mm thick) • Maximum sample volume per 10-well gel: 20 μL 25 Figure 21. Novex IEF gel migration chart using the IEF marker. Proteins shown are amyloglucosidase (Aspergillus niger), pI = 3.5; glucose oxidase (Aspergillus niger), pI = 4.2; trypsin inhibitor (soybean), pI = 4.5; β-lactoglobulin (bovine, milk), pI = 5.2 and 5.3; carbonic anhydrase (bovine, erythrocytes), pI = 6.0; myoglobin (horse, muscle), pI = 6.9 and 7.4; lectin (Lens culinaris), pI = 7.8, 8.0, and 8.3; ribonuclease A (bovine, pancreas), pI = 9.5; and cytochrome c (horse, heart), pI = 10.7. Separated on precast vertical gel (slab) Cathode (–) Anode (+) 6.9 6.0 6.0 5.3 5.2 5.3 5.2 4.5 4.2 3.5 4.5 4.2 3.5 8.3 3–10 3–7 8.0 7.8 7.4 6.9 Novex IEF gels Recommended products Invitrogen™ Novex™ IEF buffer kits include optimized cathode, anode, and sample buffers to help reduce variability and enable consistent results. IEF Marker 3–10 is ready to use and gives accurate results. ? Did you know Harry Svensson-Rilbe and his student Olof Vesterberg first described the theory of separation of amphoteric proteins along a pH gradient by applying an electric field in the 1960s. 26 Zymogram gels Easy in-gel protease analysis Novex Zymogram Plus (gelatin) gel characteristics. Zymogram Plus gelatin gel Gel composition 10% Tris-glycine gel Substrate 0.1% gelatin Sensitivity 5 x 10–6 units of collagenase Post-staining required? Yes Separation range 20–220 kDa Learn more at thermofisher.com/zymogram Specifications • Shelf life: 16 weeks • Average run time: 90 minutes • Separation range: 20–220 kDa (Figure 22) • Polyacrylamide concentrations: fixed 10% (with gelatin) • Gel dimensions: 8 x 8 cm (1 mm thick) • Maximum sample volume per well: 20 μL Invitrogen™ Novex™ 10% Zymogram Plus (gelatin) gels are useful for the detection and characterization of proteases that use gelatin as a substrate. The proteases are run under denaturing conditions and visualized as clear bands against a dark background using a simple renaturing, developing, and staining protocol. Zymogram gels are commonly used to detect matrix metalloproteases. Novex 10% Zymogram Plus gels are highly sensitive, detecting as little as 5 x 10–6 units of collagenase. Good to know How do Novex zymogram gels work? Protease samples are denatured in SDS buffer under nonreducing conditions and without heating, and run on a Novex Zymogram Plus gel in Tris-Glycine SDS Running Buffer. After electrophoresis, the proteases are renatured by incubating the gel in Invitrogen™ Novex™ Zymogram Renaturing Buffer, which contains a nonionic detergent. The gels are then equilibrated in Invitrogen™ Novex™ Zymogram Developing Buffer to add divalent metal cations required for enzymatic activity, and then stained and destained. Regions of protease activity appear as clear bands against a dark blue background where the protease has digested the substrate. 27 Figure 22. Novex Zymogram Plus (gelatin) gel migration chart. The numbered bands refer to the following proteases: Band 1: Collagenase type I (140 kDa) Band 2: Thermolysin (37 kDa) Band 3: Trypsin (19 kDa) Novex Zymogram gel 10% gel (with gelatin) 10 20 30 40 50 60 70 80 90 100 1 2 Percent length of gel 3 Recommended products After electrophoresis, incubate the gel in Novex Zymogram Renaturing Buffer (10X) to renature the enzyme. The gel is then equilibrated in Novex Zymogram Developing Buffer (10X) to add divalent metal cations required for enzymatic activity. How do E-PAGE gels work? E-PAGE gels are run in the E-Gel Power Snap Plus Electrophoresis System, which is designed to save time and streamline the protein electrophoresis workflow by enabling a simple three-step process: load, run, and analyze (Figure 23). Use of E-PAGE gels eliminates gel preparation, while preprogrammed protocols support fast and reproducible runs. Good to know 28 E-PAGE High-Throughput Precast Gel System Protein separation and analysis for increased sample throughput The Invitrogen™ E-PAGE™ High-Throughput Precast Gel System is designed for fast, bufferless medium- and high-throughput protein analysis. Invitrogen™ E-PAGE™ 48-well and 96-well precast gels consist of a buffered gel matrix and electrodes packaged inside a disposable, UV-transparent cassette. Each cassette is labeled with a unique barcode to facilitate identification of the gel using commercial barcode readers. These gels can be loaded with a multichannel pipettor or automated loading system. The E-PAGE system is compatible with the Invitrogen™ E-Gel™ Power Snap Plus Electrophoresis System, which integrates electrophoresis and real-time gel imaging into a single benchtop instrument for streamlined gel loading, running, and analysis. Advantages of using the E-PAGE High-Throughput Precast Gel System include: • Ease of use—quick setup and fast protein separation in about 23 minutes • Fast loading—compatible with multichannel pipettors and robotic loading • Efficient western blotting and staining—optimized protocols and reagents Learn more at thermofisher.com/epage Figure 23. The E-Gel Power Snap Plus Electrophoresis System offers highly reproducible results with 3 easy steps. 1 Load 2 Run 3 Analyze Specifications • Shelf life: 6 months • Average run time: 23 minutes • Separation range: 10–200 kDa (Figure 24) • Polyacrylamide concentrations: – E-PAGE 48 gel: fixed 8% – E-PAGE 96 gel: fixed 6% • Gel dimensions: 13.5 x 10.8 cm (3.7 mm thick) • Maximum sample volume per well: – E-PAGE 48 gel: 20 μL – E-PAGE 96 gel: 15 μL 29 Figure 24. E-PAGE gel migration chart. Migration patterns of the Invitrogen™ E-PAGE™ MagicMark™ Unstained Protein Standard are shown. E-PAGE gels 120 kDa 100 kDa 80 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 220 kDa 50% 10% 20% 30% 40% 60% 70% 80% 90% 0% 100% 0% 100% 75% 50% 25% E-PAGE 48 8% Gel E-PAGE 96 6% Gel 60 kDa 40 kDa 20 kDa 220 kDa 120 kDa ? Did you know Both E-PAGE 48-well and 96-well cassettes are conveniently opened with the Invitrogen™ Gel Knife (Cat. No. EI9010) to remove the gel for downstream staining or blotting. 30 SureCast Gel Handcast System 100% leak-free*—avoid pouring another gel because of leaks The Invitrogen™ SureCast™ Gel Handcast System is designed for leak-free, confident gel casting. The SureCast system is fully compatible with the Mini Gel Tank. Benefits offered by the SureCast Gel Handcast System include: • Leak-free design—less time wasted recasting after gels have leaked • Excellent glass plate durability—up to 20 times more durable than other suppliers’ plates** • Unique tilt feature—helps minimize spillage when pouring acrylamide solutions • Simple assembly of casting components—a single-motion, load-and-lock mechanism • Optional multi-use tools—all-in-one tools to guide gel loading, help open gel cassettes, and trim gels * For details, go to thermofisher.com/surecastterms. ** Based on internal testing. 31 SureCast gel handcast reagents SureCast Stacking Buffer and Resolving Buffer Invitrogen™ SureCast™ Stacking Buffer and Resolving Buffer are pouches of dry-blend powder, each sufficient to make 500 mL of stacking gel buffer (0.5 M Tris-HCl buffer, pH 6.8) or 500 mL of resolving gel buffer (1.5 M Tris buffer, pH 8.8) for handcasting polyacrylamide gels. Benefits include: • Convenient pouches of dry-blend powder—dissolve contents of a single packet in water and the buffer is ready to use • Time-and space-saving—no weighing, no calculations, no pH adjustment, and no need to stock individual components • Long shelf life—easy stocking and storage, as dry powder minimizes concerns about long-term stability of stock solutions See the SureCast Gel Handcast System in action at thermofisher.com/surecast SureCast Acrylamide Solution, 40% Invitrogen™ SureCast™ Acrylamide Solution (40%) can be used to prepare single-percentage and gradient gels using the SureCast Gel Handcast System or other handcast systems. Features include: • Room-temperature storage • Long shelf life • High purity • Safer alternative to powdered acrylamide • Concentrated to enable a broader range of gel percentages to cast 32 Good sample preparation is critical for successful separation of protein bands in electrophoresis. No single sample preparation method or buffer will work for all sample types due to the diversity of protein samples. However, the following general guidelines below should help when preparing samples for protein electrophoresis. • To minimize sample variability, keep sample preparation workflows simple, and use reagents optimized for the specific sample type and target proteins. • Cell lysis disrupts cell membranes and organelles, resulting in unregulated enzymatic activity that can reduce protein yield and lead to degraded proteins. To prevent these negative effects, protease and phosphatase inhibitors should be added to the lysis reagents. • Some buffer components may interfere with the chosen gel electrophoresis chemistry system (e.g., Tris-glycine, Bis-Tris) and cause a variety of artifacts when running the gel. Selecting a gel electrophoresis chemistry that is compatible with the buffer one’s sample is prepared in is Protein extraction and sample preparation for electrophoresis the simplest route. However, if one cannot change the gel electrophoresis chemistry system, one may need to perform sample clean-up to render the sample compatible with the given system (see “Protein clean-up methods” section). Certain clean-up methods are more favorable than others for reducing or removing specific interfering substances. • To avoid under- or overloading samples, determine the protein concentration of each sample prior to electrophoresis with a compatible protein assay (see “Protein quantitation” section). Note that certain buffer components can interfere with the chemistry of a given protein assay, and these components may need to be reduced or removed from the sample prior to determining sample concentration. These interfering substances can cause artificially high or low concentration readings, depending on the substance and the given protein assay. Cellular disruption and protein extraction Different samples (e.g., plant vs. mammalian cells) will require different disruption and extraction strategies. Use reagents that have been optimized for specific sample types. In addition, use protease and phosphatase inhibitors to minimize activity of proteases that can modify and reduce protein yields. For long-term storage of samples, we recommend freezing them to further inhibit protein degradation. Contaminant removal, desalting, concentration Remove or reduce interfering substances that can negatively impact electrophoresis. Quantitation Quantitate protein samples using a compatible protein assay. Concentrate or dilute samples if necessary. Protein sample preparation for electrophoresis 33 Obtain high protein yields from tissues, cells, or subcellular fractions using reagents and kits that are optimized for mammalian, bacterial, yeast, insect (baculovirus), and plant samples. These gentle formulations have been verified in multiple tissue types and cell lines, and generally eliminate the need for mechanical cell disruption. Protein extraction and clean-up methods Gentle formulations designed to maximize protein yield and activity Learn more at thermofisher.com/proteinextraction Overview of sample types and recommended protein extraction reagents and kits. Sample type Goal Recommended Thermo Scientific™ reagents or kits Primary cultured or mammalian cells or tissues Total protein extraction M-PER™ Mammalian Protein Extraction Reagent T-PER™ Tissue Protein Extraction Reagent N-PER™ Neuronal Protein Extraction Reagent RIPA Lysis and Extraction Buffer Pierce™ IP Lysis Buffer Cultured mammalian cells or tissues Subcellular fractionation or organelle isolation NE-PER™ Nuclear and Cytoplasmic Extraction Reagents Subcellular fractionation kits Mitochondria isolation kits Pierce™ Cell Surface Protein Isolation Kit Syn-PER™ Synaptic Protein Extraction Reagent Lysosome Enrichment Kit for Tissues and Cultured Cells Bacterial cells Total protein extraction B-PER™ Complete Bacterial Protein Extraction Reagent Yeast cells Total protein extraction Y-PER™ Yeast Protein Extraction Reagent Y-PER™ Plus Dialyzable Yeast Protein Extraction Reagent Insect cells (baculovirus) Total protein extraction I-PER™ Insect Cell Protein Extraction Reagent Plant tissue (leaf, stem, root, flower) Total protein extraction Pierce™ Plant Total Protein Extraction Kit ? Did you know Although RIPA buffer is a common lysis buffer used for preparing samples for SDS-PAGE and western blot analysis, RIPA buffer is a strong lysis buffer than contains components that may interfere with certain protein assays and gel electrophoresis chemistries. Cross reference your RIPA buffer recipe to the interfering substances lists for both gel electrophoresis and protein assays, as multiple variants on the traditional RIPA buffer recipe exist. 34 Protein electrophoresis interfering substances Interfering substance Potential issues Solution Excess salt High salt results in increased conductivity that causes uneven sample lanes and lane widening • Perform a sample clean-up method to lower salt concentration (see “Protein clean-up methods”) • Make sure that the salt concentration does not exceed 50—100 mM DNA contamination Excess DNA causes the sample to become viscous resulting in protein clumping, which can result in narrow lanes that cannot be interpreted Shear genomic DNA to reduce viscosity before loading the sample High detergent concentration Detergents can form mixed micelles with SDS and migrate through the gel. This can lead to lane widening, uneven sample lanes, and streaks in lower regions of lanes, limiting the ability to analyze proteins less than 40 kDa • Dilute the sample to reduce the final concentration of detergents in the samples loaded • Remove excess detergent with detergent removal columns or SDS-PAGE sample prep kits Sample type and protein load High protein loads can cause several issues, such as loss of protein band resolution, lane streaking, and non-straight lanes The maximum recommended sample load for optimal resolution in mini gels with 10, 12, 15, or 17 wells is 0.5 μg per band or about 10–15 μg of cell lysate per lane Excess/incorrect reducing agent Excess reducing reagent can cause shadows at lane edges The final concentration of reducing agents for SDS-PAGE should be less than 50 mM for dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), and less than 2.5% for β-mercaptoethanol (β-ME) Excess guanidine-HCl Guanidine-HCl has high ionic strength and results in increased conductivity that causes uneven sample lanes and lane widening Perform sample clean-up method Protein clean-up methods Many detergents and salts used in protein extraction formulations may have adverse effects on downstream analysis by protein electrophoresis. Therefore, it may be necessary to remove or reduce these contaminants following cell lysis or subsequent sample processing such as protein purification. Listed below are the different techniques that can be used to limit these interfering substances. Dialysis Dialysis is a classic separation technique that facilitates the removal of small, unwanted compounds from proteins in solution by selective diffusion through a semipermeable membrane. Proteins that are larger than the membrane pores are retained on the sample side of the membrane, but low molecular weight contaminants diffuse freely through the membrane and can be removed over multiple buffer exchanges. Thermo Scientific™ Slide-A-Lyzer™ dialysis cassettes and devices are ready to use and designed to minimize potential sample leakage and maximize ease of use for specific applications. Desalting Size exclusion chromatography (also known as gel filtration) can be effectively utilized for protein desalting. A resin is selected with pores that are large enough for small contaminants (e.g., salts) to penetrate, but too small for the protein of interest to enter. This causes the migration of small contaminants to slow as they get trapped in the resin, while the larger, faster proteins emerge from the column first, allowing the protein of interest to be recovered separately from the small molecules retained on the column. Thermo Scientific™ Zeba™ desalting products contain a unique resin and are specifically designed to provide consistent performance over a wide range of protein concentrations and sample sizes. High recovery of protein can be achieved even for dilute protein samples. 35 Concentration Protein concentration and diafiltration, similar to dialysis, uses a semipermeable membrane to separate macromolecules from low molecular weight compounds. Unlike dialysis, which relies on passive diffusion, concentration is achieved by forcing both liquid (buffers) and low molecular weight solutes through the membrane by centrifugation, where they are collected on the other side (filtrate). Macromolecules remain on the sample side of the membrane, where they become concentrated to a smaller volume (retentate). For buffer exchange, the retentate is diluted to the original volume with exchange buffer and centrifuged multiple times until the desired level of exchange has been achieved. Our high-performance Thermo Scientific™ Pierce™ Protein Concentrators enable rapid sample processing with high protein recovery. Comparison of protein sample clean-up methods Technique Dialysis Desalting Concentration Precipitation Best for Buffer exchange, desalting Desalting, buffer exchange Sample concentration, desalting, buffer exchange Complete buffer replacement Advantages Maximum flexibility in processing volume, wide range of molecular weight cutoffs Speed Best for low- to medium-volume samples; fast compared to traditional dialysis Low cost, good for small volumes Disadvantages Slow and multiple rounds of dialysis may be required Limited sample volume can be processed at a time Potential protein loss Higher protein loss Sample processing time ~2–24 hr ~5–10 min ~5–30 min ~5 min Sample volume processing ranges 10 μL–250 mL 2 μL–4 mL 100 μL–100 mL Any Recommended sample type Purified protein Lysate or purified protein Lysate or purified protein Lysate or purified protein Recommended product Thermo Scientific™ Slide-A-Lyzer™ Cassettes Thermo Scientific™ Zeba™ Spin Desalting Columns Thermo Scientific™ Pierce™ Protein Concentrators Thermo Scientific™ Pierce™ SDS-PAGE Sample Prep Kit Precipitation Protein precipitation removes interfering substances by selectively precipitating proteins using trichloroacetic acid (TCA) or acetone. The solution containing the interfering substance is removed, and the protein is then resolubilized in an assay‑compatible buffer. Commercially available kits simplify sample pretreatment for protein assays. Small molecules may be separated from large proteins in a sample via precipitation with acetone. When used in combination with 96-well centrifuge filter plates, this method is ideal for processing many samples at once. Download the protein clean-up handbook to learn more Good to know BCA protein assays have a unique advantage over Coomassie dye–based assays (Bradford assays), as they are compatible with samples that contain up to 5% surfactants (detergents), and are affected much less by protein compositional differences, providing greater protein-to-protein uniformity and accuracy. Tip For quantitation of low-volume or very dilute samples, fluorescent assays like the Invitrogen™ NanoOrange™ Protein Quantitation Kit can provide working ranges down to 10 ng/mL compared to 500 ng/mL for enhanced colorimetric assays and 2,000 ng/mL for standard colorimetric protocols. 36 Recommended assays based on common lysis buffers Lysis/extraction buffer Recommended protein assay If adding reducing agents or metal chelators (e.g., DTT above 1 mM, EDTA above 10 mM) RIPA Lysis and Extraction Buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) • Pierce Dilution-Free Rapid Gold BCA Protein Assay Kit • Pierce BCA Protein Assay Kit with Dilution-Free BSA Protein Standards Pierce BCA Protein Assay Kit – Reducing Agent Compatible Cell Extraction Buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton™ X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate) • Pierce BCA Protein Assay Kit – Reducing Agent Compatible Pierce BCA Protein Assay – Reducing Agent Compatible M-PER Mammalian Protein Extraction Reagent • Pierce Dilution-Free Rapid Gold BCA Protein Assay Kit • Pierce BCA Protein Assay Kit with Dilution-Free BSA Protein Standards Pierce Bradford Plus Protein Assay Kit with Dilution-Free BSA Protein Standards B-PER Bacterial Protein Extraction Reagents • Pierce Dilution-Free Rapid Gold BCA Protein Assay Kit • Pierce BCA Protein Assay Kit with Dilution-Free BSA Protein Standards Pierce BCA Protein Assay Kit – Reducing Agent Compatible NE-PER Nuclear and Cytoplasmic Extraction Reagents • Pierce Dilution-Free Rapid Gold BCA Protein Assay Kit • Pierce BCA Protein Assay Kit with Dilution-Free BSA Protein Standards Pierce Bradford Plus Protein Assay Kit with Dilution-Free BSA Protein Standards Download the technical reference guide for all available protein assays Learn more at thermofisher.com/proteinassays Protein quantitation Determining your sample’s protein concentration Protein quantitation is a necessary step before conducting analysis that compares one sample to another and to prevent under- or overloading of the gel. There are many different factors to consider when choosing the optimal protein quantification method. Two important considerations are the compatibility with common substances in the sample (detergents, reducing agents, inhibitors, salts, chaotropic agents) and the sensitivity needed. See the table below for the recommend assay based on common lysis buffers. 37 Before a sample can be loaded onto a gel for analysis, it must be properly prepared. Depending on the gel type, this may involve denaturing the proteins, reducing any disulfide bonds, and adding a sample buffer. Sample buffers contain glycerol so that they are heavier than water and sink neatly to the bottom of the buffer-submerged well when loaded onto a gel. If suitable, negatively charged, low molecular weight dye is also included in the sample buffer; it will migrate at the buffer front, enabling one to monitor the progress of electrophoresis. The most common tracking dye for sample buffers is bromophenol blue. General guidelines for preparing samples are provided below. General guidelines for preparing samples for separation: Prepare your sample in the appropriate sample buffer, sometimes referred to as loading buffer, such that the final concentration of the sample buffer is 1X. Recommended sample buffers are listed on page 38. Preparing PAGE samples for gel loading Protein assay guide Thermo Scientific™ Pierce™ Dilution-Free™ Rapid Gold BCA Protein Assay Kit Thermo Scientific™ Pierce™ BCA Protein Assay Kit with Dilution- Free™ BSA Protein Standards Thermo Scientific™ Pierce™ BCA Protein Assay Kit – Reducing Agent Compatible Thermo Scientific™ Pierce™ Bradford Plus Protein Assay Kit with Dilution-Free™ BSA Protein Standard Choose when Accuracy, reproducibility, speed, and ease of use are important Accuracy and reproducibility are important Sample contains reducing agent; accuracy and reproducibility are important Speed and ease of use are important Minimum sample volume 10 μL 25 μL 25 μL 10 μL Working range 20–10,000 μg/mL 20–2,000 μg/mL 125–2,000 μg/mL 100–1,500 μg/mL Compatibility Detergents Detergents Detergents and reducing agents Reducing agents Incubation temperature RT 37°C 37°C RT Assay incubation time 5 min 30 min 50 min 10 min Assay wavelength 480 nm 562 nm 562 nm 595 nm Running reduced and nonreduced samples: For optimal results, we do not recommend running reduced and nonreduced samples on the same gel. If you do choose to run reduced and nonreduced samples on the same gel, do not run reduced and nonreduced samples in adjacent lanes. The reducing agent may have a carry-over effect on the nonreduced samples if they are in close proximity. Heating samples: Heating the sample at 100°C in SDS-containing buffer results in proteolysis [3]. We recommend heating samples for denaturing electrophoresis (reduced or nonreduced) at 70°C for 2–10 minutes for optimal results. Do not heat the samples for nondenaturing (native) electrophoresis or Invitrogen™ Novex™ Zymogram Plus gels. 38 Learn more at thermofisher.com/ electrophoresisbuffers Electrophoresis buffers We offer reliable premixed SDS-PAGE buffers and reagents, including sample buffers, running buffers, reducing agents, and antioxidants. Reducing agent When preparing samples for reducing gel electrophoresis, any of the following reducing agents may be used: • Bolt Sample Reducing Agent • NuPAGE Sample Reducing Agent • Dithiothreitol (DTT), 50 mM final concentration • β-mercaptoethanol (β-ME), 2.5% final concentration • Tris-(2-carboxyethyl)phosphine (TCEP), 50 mM final concentration Add the reducing agent to the sample up to an hour before loading the gel. Avoid storing reduced samples for long periods, even if they are frozen. Reoxidation of samples can occur during storage and produce inconsistent results. MES vs. MOPS running buffer • Use MES SDS running buffers to resolve small molecular weight proteins. • Use MOPS running buffers to resolve medium-sized proteins. • MES has a lower pKa than MOPS, enabling gels with MES running buffer to run faster than gels with MOPS SDS running buffer. The difference in ion migration affects stacking and results in a difference in protein separation range between these buffers. Buffer and reagent selection guide Gel type Sample buffers optimized for use with the gel Running buffers optimized for use with the gel Bolt Bis-Tris Plus gel • Bolt Sample Reducing Agent (10X) • 4X Bolt LDS Sample Buffer (nonreducing) • Bolt Antioxidant • 20X Bolt MES SDS Running Buffer (3.5–160 kDa separations) • 20X Bolt MOPS SDS Running Buffer (15–260 kDa separations) NuPAGE Bis-Tris gel • NuPAGE Sample Reducing Agent (10X) • 4X NuPAGE LDS Sample Buffer (nonreducing) • NuPAGE Antioxidant • NuPAGE MES SDS Running Buffer (20X liquid or powder) (3.5–160 kDa separations) • NuPAGE MOPS SDS Running Buffer (20X liquid or powder) (15–260 kDa separations) NuPAGE Tris-Acetate gel • 4X NuPAGE LDS Sample Buffer (nonreducing) • NuPAGE Sample Reducing Agent (10X) • Novex Tris-Glycine Native Sample Buffer (2X)* • NuPAGE Tris-Acetate SDS Running Buffer (20X) • Novex Tris-Glycine Native Running Buffer (10X)* Novex Tris-Glycine gel • Novex Tris-Glycine SDS Sample Buffer (2X) • NuPAGE Sample Reducing Agent (10X) • Novex Tris-Glycine Native Sample Buffer (2X)* • Novex Tris-Glycine SDS Running Buffer (10X) • Novex Rapid Tris-Glycine SDS Running Buffer (10X liquid or powder) • Novex Tris-Glycine Native Running Buffer (10X)* • Pierce Tris-Glycine SDS Buffer (10X) • BupH Tris-Glycine Buffer Packs Novex Tricine gel • Novex Tricine SDS Sample Buffer (2X) • Novex Tricine SDS Running Buffer (10X) NativePAGE gel • NativePAGE Sample Buffer (4X) • NativePAGE 5% G-250 Sample Additive • NativePAGE Running Buffer (20X) • NativePAGE Cathode Buffer Additive (20X) Novex IEF gel • Novex IEF Sample Buffer, pH 3–10 (2X) • IEF Sample Buffer, pH 3–7 (2X) • Novex IEF Anode Buffer (50X) • Novex IEF Cathode Buffer, pH 3–10 (10X) • Novex IEF Cathode Buffer, pH 3–7 (10X) Novex Zymogram Plus gel** • Novex Tris-Glycine SDS Sample Buffer (2X) • Novex Tris-Glycine SDS Running Buffer (10X) * Use this buffer when performing a native separation with this gel. ** Novex Zymogram Developing Buffer (10X) and Novex Zymogram Renaturing Buffer (10X) are available for visualizing the Zymogram Plus gels. 39 Buffer recipes Bolt Bis-Tris buffer recipes Buffer Storage Component Concentration (1X) Bolt LDS Sample Buffer 4–25°C Glycerol Tris base Tris-HCl LDS EDTA SERVA™ Blue G-250 Phenol red 10% 141 mM 106 mM 2% 0.51 mM 0.22 mM 0.175 mM (pH 8.5) Bolt MOPS SDS Running Buffer 4–25°C MOPS Tris base SDS EDTA 50 mM 50 mM 0.1% 1 mM (pH 7.7) Bolt MES SDS Running Buffer 4–25°C MES Tris base SDS EDTA 50 mM 50 mM 0.1% 1 mM (pH 7.3) Bolt Transfer Buffer 4–25°C Bicine Bis-Tris (free base) EDTA Chlorobutanol 25 mM 25 mM 1.0 mM 0.05 mM (pH 7.2) 40 NuPAGE Bis-Tris and Tris-acetate buffer recipes Buffer Storage Component Concentration (1X) NuPAGE LDS Sample Buffer 4–25°C Glycerol Tris base Tris-HCl LDS EDTA SERVA Blue G-250 Phenol red 10% 141 mM 106 mM 2% 0.51 mM 0.22 mM 0.175 mM (pH 8.5) NuPAGE MOPS SDS Running Buffer* 4–25°C MOPS Tris base SDS EDTA 50 mM 50 mM 0.1% 1 mM (pH 7.7) NuPAGE MES SDS Running Buffer* 4–25°C MES Tris base SDS EDTA 50 mM 50 mM 0.1% 1 mM (pH 7.3) NuPAGE Tris-Acetate SDS Running Buffer 4–25°C Tris base Tricine SDS 50 mM 50 mM 0.1% (pH 8.24) NuPAGE Transfer Buffer 4–25°C Bicine Bis-Tris (free base) EDTA Chlorobutanol 25 mM 25 mM 1.0 mM 0.05 mM (pH 7.2) * The premixed buffers (Cat. Nos. NP0001 and NP0002) also contain trace amounts of the proprietary NuPAGE Antioxidant (Cat. No. NP0005) for stability. Additional antioxidant may be required with specific protocols. Novex Tris-Glycine buffer recipes Buffer Storage Component Concentration (1X) Novex Tris-Glycine SDS Sample Buffer 4°C Tris-HCl* Glycerol SDS Bromophenol blue Deionized water 63 mM 10% 2% 0.0025% —( pH 6.8) Novex Tris-Glycine Native Sample Buffer 4°C Tris-HCl* Glycerol Bromophenol blue Deionized water 100 mM 10% 0.0025% —( pH 8.6) Novex Tris-Glycine SDS Running Buffer Room temperature Tris base Glycine SDS Deionized water 25 mM 192 mM 0.1% —( pH 8.3) Novex Tris-Glycine Native Running Buffer Room temperature Tris base Glycine Deionized water 25 mM 192 mM —( pH 8.3) Novex Tris-Glycine Transfer Buffer Room temperature Tris base Glycine Deionized water 12 mM 96 mM —( pH 8.3) * Tris-HCl solutions are prepared from Tris base and pH-adjusted with 6 N HCl. 41 Novex Tricine buffer recipes Buffer Storage Component Concentration (1X) Novex Tricine SDS Sample Buffer +4˚C Tris-HCl* Glycerol SDS Coomassie Blue G Phenol red Deionized water 450 mM 12% 4% 0.0075% 0.0025% —( pH 8.45) Novex Tricine SDS Running Buffer Room temperature Tris base Tricine SDS Deionized water 100 mM 100 mM 0.1% —( pH 8.3) * Tris-HCl solutions are prepared from Tris base and pH-adjusted with 6 N HCl. Zymogram buffer recipes Buffer Storage Component Concentration (1X) Novex Zymogram Plus Renaturing Buffer Room temperature Triton X-100 solution Deionized water 2.7% (w/v) in H2O — Novex Zymogram Plus Developing Buffer Room temperature Tris-HCI* NaCl CaCl2·2H2O Brij 35 Deionized water 50 mM 200 mM 5 mM 0.006% (w/v) —( pH 7.6) * Tris-HCl solutions are prepared from Tris base and pH-adjusted with 6 N HCl. Isoelectric focusing buffer recipes Buffer Storage Component Concentration (1X) Novex IEF Sample Buffer pH 3–7 4°C Lysine (free base) Glycerol Deionized water 40 mM 15% — Novex IEF Sample Buffer pH 3–10 4°C Arginine (free base) Lysine (free base) Glycerol Deionized water 20 mM 20 mM 15% — Novex IEF Cathode Buffer pH 3–7 (upper buffer chamber) 4°C Lysine (free base) Deionized water 40 mM — Novex IEF Cathode Buffer pH 3–10 (upper buffer chamber) 4°C Arginine (free base) Lysine (free base) Deionized water 20 mM 20 mM —( pH 10.1) Novex IEF Anode Buffer (for both pH ranges) (lower buffer chamber) Room temperature Phosphoric acid 85% Deionized water 7 mM —( pH 2.4) Urea-thiourea-CHAPS (rehydration buffer for IPG strips) –20°C Deionized urea Deionized thiourea CHAPS Ampholytes Bromophenol blue Ultrapure water DTT 7 M 2 M 2–4% 0.2–2.0% 0.002% — 20 mM Estimating protein sizes Protein ladders and standards 42 Protein ladders, also known as protein markers or protein standards, are used to help estimate the size of proteins separated during electrophoresis. They serve as points of reference because they contain mixtures of highly purified proteins with known molecular weights and characteristics. Protein ladders are loaded onto gels alongside samples and migrate during electrophoresis at a rate that is inversely proportional to their molecular sizes. When the run is complete, the proteins will appear as separate bands in the gel. A standard curve can be constructed from the distance each marker protein migrates through the gel versus the log of its molecular weight. After measuring the migration distance that an unknown protein travels through the same gel, its molecular weight can be determined from the standard curve. Modern gel documentation instruments may have software algorithms that can make these mathematical calculations significantly more convenient. More often, however, protein ladders are used as a reference to help confirm the identity of proteins of interest whose sizes are already known. Protein ladders are available with various protein molecular weight ranges and can be prestained, unstained, or labeled for different modes of detection and downstream applications. Read on to learn which protein ladder is best for your applications. Learn more at thermofisher.com/proteinladders 43 Prestained protein ladders Broad range High range . Thermo Scientific™ PageRuler™ Plus Prestained Protein Ladder, 10–250 kDa Invitrogen™ SeeBlue™ Prestained Protein Standard, 3–198 kDa Thermo Scientific™ Spectra™ Multicolor High Range Protein Ladder, 10–260 kDa Invitrogen™ HiMark™ Prestained Protein Standard 31–460 kDa Cat. No. 26619 Cat. No. LC5625 Cat. No. 26625 Cat. No. LC5699 Prestained protein ladders contain proteins prelabeled with various dyes that allow them to be visible without additional staining. Prestained ladders are visible during electrophoresis, enabling the ability to monitor the progress of separation for the range of protein sizes that are most relevant to the particular experiment. Prestained markers and ladders also remain visible after gel staining or transfer to membranes for detection by western blotting. Prestained protein ladders can be used to: • Monitor protein separation during polyacrylamide gel electrophoresis • Quickly check visually whether protein transfer has occurred from gel to membrane (prestained protein ladder should transfer to membrane but should not be used to determine transfer efficiency; see “Good to know” notes) • Estimate the approximate size of your target protein 44? Why do molecular weights change between the different gel chemistries? Slight differences in protein mobilities will occur when the same proteins are run in different SDS-PAGE buffer systems (e.g., Bis-Tris vs. Tris-glycine). Each SDS-PAGE buffer system has a different pH, which affects the charge of a protein and its binding capacity for SDS. The degree of change in protein mobility is usually small in natural proteins but is more pronounced with atypical or chemically modified proteins, such as prestained standards. Apparent molecular weight values for prestained standards will vary between gel systems—it is important to use the apparent molecular weights that match your gel for the most accurate calibration of your sample proteins. Good to know How can protein transfer efficiency and consistency be assessed? Transfer efficiency refers to the efficiency of transfer of protein out of the protein gel and onto the western blotting membrane. Specifically, the amount of protein that binds to the membrane will be the foundation for the immunoblotting steps. If very little protein transfers out of the gel and binds to the membrane, then the subsequent immunoblotting steps will be challenging. Transfer efficiency is sometimes evaluated by visually assessing the amount of prestained protein ladder that is visible on the membrane post-transfer. However, this is not necessarily the best practice. Prestained ladder proteins are stained with dyes that allow for visualization during electrophoresis. These dyes affect the transfer of the ladder proteins out of the gel. Sample proteins, however, are not stained and thus can have a different mobility rate out of the gel. Visual assessment of the transfer of ladder can be a useful quick process check for whether transfer occurred (sometimes electrodes may be reversed, or the transfer stack may not be assembled correctly). Not observing any prestained marker transfer to the membrane could indicate an issue with the transfer setup. A more favorable strategy to determine transfer efficiency is to use a reversible membrane stain, which will stain the total protein that successfully transferred to the membrane. Some choose to take this a step further and back-stain the gel, post-transfer, which can give an assessment of how much protein transferred out of the gel. It is not unusual to see ladder protein or sample protein remaining in the gel, as no transfer method is completely 100% efficient. What really matters is how much protein is bound to the membrane. Transfer consistency refers to the evenness of protein transfer from gel to membrane. Ideally, protein is transferred evenly from side to side and top to bottom on the membrane. However, improper assembly of a transfer stack and failure to roll the transfer stack may lead to air bubbles, creases, and dead spots on the membrane. These artifacts make the subsequent immunoblotting steps challenging, as an uneven transfer of protein will lead to an uneven immunoblot and signal production. Transfer consistency cannot be assessed simply by the appearance of transferred prestained ladder proteins on the membrane. Instead, the use of a reversible membrane stain permits a more accurate picture of protein transfer efficiency across the entire membrane. Good to know 45 Western blot protein ladders Western blot–specific protein ladders are designed for easy and convenient protein molecular weight estimation directly on western blots or indirectly by using various blotting detection systems (e.g., chemiluminescent, fluorescent, or chromogenic). The protein markers consist of recombinant proteins with an IgG binding site. The IgG binding site binds the primary or secondary antibody used for detection of the target protein, allowing visualization of the standard on the western blot. Recommended for: • Approximate molecular weight determination directly on western blots or indirectly through chemiluminescent, fluorescent, or chromogenic detection systems • Qualitatively assessing transfer efficiency Western blot Invitrogen™ iBright™ Prestained Protein Ladder, 11–250 kDa Invitrogen™ MagicMark™ XP Western Protein Standard, 20–220 kDa Cat. No. LC5615 Cat. No. LC5602 46 Unstained protein ladders Unstained protein ladders are useful when you need to accurately determine the size of your protein. However, the proteins can only be visualized after staining with a Coomassie stain or other nonspecific protein stain. The dyes in prestained protein ladders add additional molecular weight to the ladder proteins and thus affect their migration. Each dye molecule may not bind equally to a given protein or proteins, and some prestained protein ladders utilize multiple different types of colored dyes (for easier tracking of specific proteins in the ladder). Because of the dyes in prestained protein ladders, a given ladder protein’s true molecular weight is altered and thus is an apparent molecular weight. Unstained protein ladders do not have this issue because the migration of the proteins in the ladder are not affected by added dye, and thus migrate as normal (although one can observe differences from one gel chemistry to the next), and as such are suitable for the most accurate determination of molecular weights in a protein sample. These ready-to-use ladders include proteins of known molecular weight. Visualize them on SDS-PAGE gels by staining with Coomassie Brilliant Blue R250 or after western transfer using Ponceau S or a similar stain. High range Broad range Broad range 8–16% Tris-glycine gel (SDS-PAGE) kDa –150– –120– –100– —85— —70— —60— —50— —40— —30— —25— —20— —15— —10— Gel Blot –200– Invitrogen™ HiMark™ Unstained Protein Standard Thermo Scientific™ PageRuler™ Unstained Protein Ladder Thermo Scientific™ PageRuler™ Unstained Broad Range Protein Ladder Cat. No. LC5688 Cat. No. 26614 Cat. No. 26630 47 IEF and specialty protein ladders IEF His-tagged proteins Phosphorylated proteins Glycosylated proteins pl 8.0 6.9 8.3 9.5 10.7 4.5 4.2 3.5 5.3 6.0 5.2 7.4 7.8 Gel: Invitrogen™ Novex™ pH 3–10 IEF Protein Gel SimplyBlue stain InVision stain — 116 — 66 — 45 — 24 — 18 kDa — 14 Invitrogen™ IEF Marker 3–10 Invitrogen™ BenchMark™ His-Tagged Protein Standard Invitrogen™ PeppermintStick™ Phosphoprotein Molecular Weight Standards Invitrogen™ CandyCane™ Glycoprotein Molecular Weight Standards Cat. No. 39212-01 Cat. No. LC5606 Cat. No. P33350 Cat. No. C21852 Uses: IEF applications Uses: positive control and for molecular weight sizing in His-tagged fusion protein detection Uses: positive and negative controls for detection of phosphorylated proteins Uses: positive and negative controls for detection of glycosylated proteins Visualization: colorimetric stain Visualization: Invitrogen™ InVision™ His-Tag In-Gel Stain or Invitrogen™ 6x-His Tag Monoclonal Antibody Visualization: colorimetric stain or methods that detect phosphorylated proteins, such as Invitrogen™ Pro-Q™ Diamond phosphoprotein gel stains Visualization: colorimetric stain or methods that detect glycosylated proteins, such as Invitrogen™ Pro-Q™ Emerald glycoprotein stain kits Protein ladders are available for use in isoelectric focusing (IEF) and for use when the experiment involves the detection of His-tagged, phosphorylated, or glycosylated proteins. 48 In electrical terms, the process of electrophoresis is closely associated with the following equations derived from Ohm’s law: Voltage = current × resistance (V = IR) Wattage = current × voltage (W = IV) Resistance The electrical resistance of the assembled electrophoresis cell is dependent on buffer conductivity, gel thickness, temperature, and the number of gels being run. Although the resistance is determined by the gel system, the resistance varies over the course of the run. • In discontinuous buffer systems (and to a lesser extent in continuous buffer systems) resistance increases over the course of electrophoresis. This occurs in the Tris-glycine buffer system as highly conductive chloride ions in the gel are replaced by less conductive glycine ions from the running buffer. • Resistance decreases as the temperature increases. Voltage The velocity of an ion in an electric field varies in proportion to the field strength (volts per unit distance). The higher the voltage, the faster an ion moves. For most applications, we recommend a constant voltage setting. • A constant voltage setting allows the current and power to decrease over the course of electrophoresis, providing a safety margin in case of a break in the system. • The constant voltage setting does not need adjustment to account for differences in number or thickness of gels being electrophoresed. Choosing the electrophoresis chamber and power supply Current For a given gel/buffer system, at a given temperature, current varies in proportion to the field strength (voltage) and cross‑sectional area (thickness and number of gels). When using a constant current setting, migration starts slow, and accelerates over time, thus favoring stacking in discontinuous gels. When running under constant current, set a voltage limit on the power supply at or slightly above the maximum expected voltage to avoid unsafe conditions. At constant current, voltage increases as resistance increases. If a local fault condition occurs (e.g., a bad connection), high local resistance may cause the voltage to reach the maximum for the power supply, leading to overheating and damage of the electrophoresis cell. Power Wattage measures the rate of energy conversion, which is manifested as heat generated by the system. Using constant power allows the total amount of heat generated by the system to remain constant throughout the run, but results in variable mobility since voltage increases and current decreases over the course of the run. Constant power is typically used when running IEF strips. When using constant power, set the voltage limit slightly above the maximum expected for the run. High local resistance can cause a large amount of heat to be generated over a small distance, damaging the electrophoresis cell and gels. 49 Mini Gel Tank XCell SureLock Mini-Cell SureLock Tandem Midi Gel Tank XCell4 SureLock Midi-Cell Tetra Electrophoresis Cores Gel capacity Up to 2 mini gels Up to 2 mini gels Up to 2 midi gels Up to 4 midi gels Up to 4 mini gels Cell dimensions (L x W x H; height with lid on) 32 x 11.5 x 16 cm 14 x 13 x 16 cm 25 × 17.9 × 17.3 cm 21 x 19 x 16 cm Advantages • The Mini Gel Tank is versatile and compatible with all Invitrogen precast and handcast mini gels; the unique tank design enables convenient side-by-side gel loading and enhanced viewing during use • Mini Blot Module is available for wet protein transfers • Instrument incorporates a gel tension wedge in place of the rear wedge used on earlier models • XCell II Blot Module is available for semi-wet protein transfers • Easy-to-use apparatus, with separate chambers for each gel, enabling scalable buffer usage • SureLock Tandem Midi Blot Module is available for wet protein transfers • Advanced apparatus for easier, more reliable electrophoresis with midi gels • Tetra Electrophoresis Cores allow high-performance Invitrogen precast gels to be run in the Bio-Rad Mini- PROTEAN Tetra Cell tank • Easy to assemble and use with one or up to 4 mini gels Learn more at thermofisher.com/electrophoresischambers Electrophoresis chamber system selection guide Our electrophoresis chamber systems are designed for compatibility with the full range of Invitrogen gel offerings. Refer to the table below to find which system is right for you. 50 Mini Gel Tank One tank, over 150 gels The Mini Gel Tank is designed for more intuitive use and greater convenience compared to traditional electrophoresis tanks. The unique, side-by-side tank design allows you to perform electrophoresis of 1 or 2 mini gels. Learn more at thermofisher.com/minigeltank Specifications • Gel capacity: up to 2 mini gels • Cell size (L x W x H): 32 x 11.5 x 16 cm (height with lid on) • Buffer requirement: 400 mL for each mini gel chamber • Material: polycarbonate • Chemical resistance: not compatible with acetone, chlorinated hydrocarbons (e.g., chloroform), or aromatic hydrocarbons (e.g., toluene, benzene) Watch our Mini Gel Tank video The Mini Gel Tank offers: • Versatility—compatible with all of our mini gels, including Invitrogen™ NuPAGE™, Novex™, Bolt™, and specialty gels • Easy sample loading—forward-facing well configuration • Simultaneous visualization of both gels—streamlined, side-by-side tank configuration • Simple monitoring of gels—white tank stand permits easy visualization of prestained markers • Less running buffer required—gel chambers are separated, so you only need to load sufficient buffer for each gel to the specified fill line • Engineered to stand the test of time—made of durable polycarbonate, it undergoes a rigorous stress-relieving process that helps prevent cracking 51 Figure 25. How to use the Mini Gel Tank. Figure 26. Electrophoresis of Bolt gel using the Mini Gel Tank. Protein standards and samples were loaded at 10 μL sample volumes on an Invitrogen™ Bolt™ 4–12% Bis-Tris Plus gel. Electrophoresis was performed using the Mini Gel Tank at 200 V (constant). Sharp, straight bands with consistent migration patterns were observed after staining with Invitrogen™ SimplyBlue™ SafeStain. Images were acquired using a flatbed scanner. Lane 1: SeeBlue Plus2 Prestained Standard; lane 2: 10 μg E. coli lysate; lane 3: Invitrogen™ Mark12™ Unstained Standard (blend of 12 purified proteins); lane 4: 40 μg HeLa cell lysate; lane 5: 20 μg HeLa cell lysate; lane 6: 5 μg BSA; lane 7: 40 μg Jurkat cell lysate; lane 8: 5 μg GST fusion protein; lane 9: Invitrogen™ Novex™ Sharp Unstained Protein Standard; lane 10: 5 μg β-galactosidase. Recommended products The Invitrogen™ Mini Blot Module is a wet transfer apparatus that conveniently fits into the chambers of the Mini Gel Tank to easily transfer proteins from mini gels to nitrocellulose or PVDF membranes. 1. Snap the electrophoresis tank into the base, and place the cassette clamp(s) into the chamber(s) with the anode connector(s) (+) aligned to the center. Fill the chamber(s) with 1X buer to the level of the cathode. 4. Make sure the wells are completely filled with 1X buer. Load your samples and markers. 2. Remove the comb, and peel away the tape at the bottom of the gel cassette. Rinse the wells 3 times with 1X buer. 5. Hold the cassette and release the cassette clamp. Gently lower the casette so that it rests on the bottom of the chamber, and close the cassette clamp. Add 1X buer to the level of the fill line. 6. Make sure the power supply is o. If only running one gel, remove the cassette clamp from unused chamber. Place the lid on the tank and plug the electrode cords into the power supply. Turn the power supply on to begin electrophoresis. 3. Place the cassette in the chamber with the wells facing towards you. Hold the cassette in a raised position and close the clamp by moving the cam handle forward. the base, and the chamber(s) aligned to the center. to the level of completely filled with 2. Remove the comb, and peel away the tape at the bottom of the gel cassette. Rinse the wells 3 times with 1X buer. 5. Hold the cassette and release the cassette clamp. Gently lower the casette so that it rests on the bottom of the chamber, and close the cassette clamp. Add 1X buer to the level of the fill line. 6. Make sure the power supply is o. If only running one gel, remove the cassette clamp from unused chamber. Place the lid on the tank and plug the electrode cords into the power supply. Turn the power supply on to begin electrophoresis. 3. Place the cassette in the chamber with the wells facing towards you. Hold the cassette in a raised position and close the clamp by moving the cam handle forward. the tape at the er. cassette clamp. rests on the the cassette clamp. line. 6. Make sure the power supply is o. If only running one gel, remove the cassette clamp from unused chamber. Place the lid on the tank and plug the electrode cords into the power supply. Turn the power supply on to begin electrophoresis. 3. Place the cassette in the chamber with the wells facing towards you. Hold the cassette in a raised position and close the clamp by moving the cam handle forward. 1. Snap the electrophoresis tank into the base, and place the cassette clamp(s) into the chamber(s) with the anode connector(s) (+) aligned to the center. Fill the chamber(s) with 1X buer to the level of the cathode. 4. Make sure the wells are completely filled with 1X buer. Load your samples and markers. 2. Remove the comb, and peel away the tape at the bottom of the gel cassette. Rinse the wells 3 times with 1X buer. 5. Hold the cassette and release the cassette clamp. Gently lower the casette so that it rests on the bottom of the chamber, and close the cassette clamp. Add 1X buer to the level of the fill line. 6. Make sure the power supply is o. If only running one gel, remove the cassette clamp from unused chamber. Place the lid on the tank and plug the electrode cords into the power supply. Turn the power supply on to begin electrophoresis. 3. Place the cassette in the chamber with the wells facing towards you. Hold the cassette in a raised position and close the clamp by moving the cam handle forward. the base, and the chamber(s) aligned to the center. to the level of completely filled with 2. Remove the comb, and peel away the tape at the bottom of the gel cassette. Rinse the wells 3 times with 1X buer. 5. Hold the cassette and release the cassette clamp. Gently lower the casette so that it rests on the bottom of the chamber, and close the cassette clamp. Add 1X buer to the level of the fill line. 6. Make sure the power supply is o. If only running one gel, remove the cassette clamp from unused chamber. Place the lid on the tank and plug the electrode cords into the power supply. Turn the power supply on to begin electrophoresis. 3. Place the cassette in the chamber with the wells facing towards you. Hold the cassette in a raised position and close the clamp by moving the cam handle forward. the tape at the er. cassette clamp. rests on the the cassette clamp. line. 6. Make sure the power supply is o. If only running one gel, remove the cassette clamp from unused chamber. Place the lid on the tank and plug the electrode cords into the power supply. Turn the power supply on to begin electrophoresis. 3. Place the cassette in the chamber with the wells facing towards you. Hold the cassette in a raised position and close the clamp by moving the cam handle forward. 1. Snap the electrophoresis tank into the base, and place the cassette clamp(s) into the chamber(s) with the anode connector(s) (+) aligned to the center. Fill the chamber(s) with 1X buffer to the level of the cathode. 2. Remove the comb, and peel away the tape at the bottom of the gel cassette. Rinse the wells 3 times with 1X buffer. 3. Place the cassette in the chamber with the wells facing towards you. Hold the cassette in a raised position and close the clamp by moving the cam handle forward. 4. Make sure the wells are completely filled with 1X buffer. Load your samples and markers. 5. Hold the cassette and release the cassette clamp. Gently lower the casette so that it rests on the bottom of the chamber, and close the cassette clamp. Add 1X buffer to the level of the fill line. 6. Make sure the power supply is off. If only running one gel, remove the cassette clamp from unused chamber. Place the lid on the tank and plug the electrode cords into the power supply. Turn the power supply on to begin electrophoresis. 52 The unique design of the Invitrogen™ XCell SureLock™ Mini-Cell allows you to run mini gels quickly and easily without any clamps or grease (Figure 27). The tight seal provided by the gel tension wedge results in consistent performance. The XCell SureLock Mini-Cell is compatible with Bolt, NuPAGE, Novex, and specialty gels (Figure 28). XCell SureLock Mini-Cell Simultaneous electrophoresis of up to 2 mini gels Specifications • Gel capacity: up to 2 mini gels • Cell size (L x W x H): 14 x 13 x 16 cm (height with lid on) • Buffer chamber requirement (Invitrogen mini gels): – Upper buffer chamber: 200 mL – Lower buffer chamber: 600 mL • Chemical resistance: impervious to most alcohols but not compatible with acetone, chlorinated hydrocarbons (e.g., chloroform), or aromatic hydrocarbons (e.g., toluene, benzene) Key features of the XCell SureLock Mini-Cell: • User-friendly design—uses single gel tension wedge with no clamps or grease • Flexibility—perform electrophoresis of 2 mini gels simultaneously • Unique, heat dissipating design—no need for a cooling device • Built-in usability features—retractable plugs, recessed jacks, and a specially designed lid enhances user safety Figure 28. Electrophoresis of NuPAGE Bis-Tris gel with the XCell SureLock Mini-Cell. Lane 1: SeeBlue Plus2 Prestained Standard; lane 2: 10 μg E. coli lysate; lane 3: Mark12 Unstained Standard (blend of 12 purified proteins); lane 4: 40 μg HeLa cell lysate; lane 5: 20 μg HeLa cell lysate; lane 6: not used; lane 7: 40 μg Jurkat cell lysate; lane 8: 5 μg of a GST fusion protein; lane 9: Novex Sharp Unstained Protein Standard; lane 10: 5 μg β-galactosidase. Gel electrophoresis was performed at 200 V (constant), and gels were stained using SimplyBlue SafeStain. Image was acquired using a flatbed scanner. Recommended products The XCell SureLock Mini-Cell can be easily adapted for transfer of proteins from mini gels to membranes by simply inserting the Invitrogen™ XCell II™ Blot Module into the lower buffer chamber. NuPAGE Bis-Tris gel in XCell SureLock Mini-Cell Learn more at thermofisher.com/surelockmini 1. Drop buffer core into the lower buffer chamber of the XCell SureLock Mini- Cell. Insert one mini gel in front of the buffer core and a second mini gel or the buffer dam behind the buffer core. 2. Lock the gel tension wedge in place, load samples, and fill the buffer chambers with the appropriate running buffers. 3. Place the cell lid on the unit and you’re ready to run. Figure 27. How to use the XCell SureLock Mini-Cell. 53 The Invitrogen™ SureLock™ Tandem Midi Gel Tank enables rapid electrophoresis of midi gels using minimal buffer (~520 mL/gel) in a leak-free system. With a setup time of ~30 seconds, the tank efficiently runs midi gels while also providing consistent performance. The tank features two independent chambers, allowing electrophoresis of one or two gels at a time, which saves on buffer and limits waste. The SureLock Tandem Midi Gel Tank can be used for wet tank transfers when paired with the SureLock Tandem Midi Blot Module. The SureLock Tandem Midi Blot Module performs efficient, room-temperature wet protein transfers for downstream western blot analysis. The tank accommodates two blot modules, allowing transfer of one or two gels at a time, using considerably less transfer buffer (only ~300 mL per transfer) than other wet transfer systems. This lower buffer requirement keeps the amount of methanol waste (a hazardous material) to a minimum. SureLock Tandem Midi Gel Tank 2-in-1 midi gel electrophoresis and transfer tank View performance data and a how-to video at thermofisher.com/surelocktandem Figure 29. Publication-quality protein electrophoresis gel results using a NuPAGE Bis-Tris 4–12% gradient midi gel and the SureLock Tandem Midi Gel Tank. The gel was loaded as follows: Lanes 1, 20: 5 μL PageRuler Broad Range Unstained Protein Ladder (Cat. No. 26630); lanes 2–7: 10 μg, 8 μg, 6 μg, 4 μg, 2 μg, 1 μg HeLa lysate; lanes 8, 13: 5 μL Novex Mark12 Unstained Standard (Cat. No. LC5677); lanes 9–12: 240 ng, 180 ng, 120 ng, 60 ng of protein mix containing β-galactosidase, lactate dehydrogenase, and lysozyme; lanes 14–19: 10 μg, 8 μg, 6 μg, 4 μg, 2 μg, 1 μg E. coli lysate. Electrophoresis was conducted with NuPAGE MOPS running buffer and stained with SimplyBlue Safe Stain. NuPAGE Bis-Tris 4–12% gradient midi gel Specifications • Gel capacity: up to 2 midi gels (8 x 13 mm) • Cell size (L x W x H): 25 x 17.9 x 17.3 cm (height with lid on) • Buffer chamber requirement: – Upper chamber: 170 mL (per gel) – Lower chamber: 350 mL (per gel) • Chemical resistance: impervious to most alcohols but not compatible with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene, benzene), acetone, or isopropyl alcohol Key features of the SureLock Tandem Midi Gel Tank: • Double duty instrument—enables electrophoresis and transfer of high-performance Invitrogen™ midi gels using the same tank • Two separate chambers—run 1 or 2 gels or transfers using only the necessary amount of buffer for each gel, minimizing buffer cost and waste • Room-temperature transfer—eliminate the need to prechill buffers and the hassle and messiness of ice baths • User-friendly design—easy setup, sample loading, and simple workflow, without dummy cassettes or buffer dams • Compatible—fits all Invitrogen precast midi gels and Invitrogen midi gel cassettes 54 The Invitrogen™ XCell4 SureLock™ Midi-Cell allows simultaneous electrophoresis of 1–4 midi gels without leaking, enabling consistent performance. The system is designed to dissipate heat effectively and evenly, and enable high-resolution results when using Invitrogen™ Novex™ midi gels (Figures 30 and 31). Key features of the XCell4 SureLock Midi-Cell: • User-friendly design—electrophoresis without clamps or grease • Flexibility—perform electrophoresis of 1–4 midi gels • Unique, heat-dissipating design—no need for a cooling device • Built-in safety features—specially designed lid enhances safety XCell4 SureLock Midi-Cell Simultaneous electrophoresis of up to 4 midi gels Specifications • Gel capacity: up to 4 midi gels (8 x 13 cm) • Cell size (L x W x H): 21 x 19 x 16 cm (height with lid on) • Buffer chamber requirement: – Upper buffer chamber: 175 mL x 4 – Lower buffer chamber: 540–700 mL • Chemical resistance: not compatible with acetone, chlorinated hydrocarbons (e.g., chloroform), or aromatic hydrocarbons (e.g., toluene, benzene) 55 Learn more at thermofisher.com/surelockmidi 1. Insert the XCell4 SureLock Midi-Cell assembly in its unlocked position into the center of the midi-cell base. The XCell4 SureLock assembly slides down over the protrusion in the midi-cell base. 2. Place one cassette on each side of the buffer core for each of the two cores. For each cassette, the shorter “well” side of the cassette must face out towards the lower buffer chamber. 3. While holding the assembly together with your hands (A), insert the buffer cores with the gel cassettes into the lower buffer chamber such that the negative electrode fits into the opening in the gold plate on the lower buffer chamber (B). Always hold the cassette assembly by its edges as shown in the figure. Note: If you are having difficulty inserting the assembly into the lower buffer chamber, make sure the cathode (black polarity indicator) of the buffer core is aligned with the cathode (black polarity indicator) of the lower buffer chamber. 4. The upper buffer chamber (cathode) is the void formed between a gel and the buffer core at the center of each core. 5. Lock the XCell4 SureLock assembly by moving the tension lever to the locked position (indicated on the XCell4 SureLock Assembly). This will squeeze the gels and buffer cores together, creating leak-free seals. 6. Proceed to loading samples and buffers. A B Figure 31. Quality of a precast Invitrogen™ NuPAGE™ 4–12% Bis-Tris Midi Gel with a variety of protein standards, lysates, and purified proteins. Lanes 1, 10, 11, 20: 5 μL of Mark12 Unstained Standard (blend of 12 purified proteins); lanes 2, 9, 12, 19: 10 μg of E. coli lysate; lanes 3, 18: 6 μg of human IgG; lanes 4, 17: 6 μg of human IgM; lanes 5, 16: 5 μL of SeeBlue Plus2 Prestained Protein Standard; lanes 6, 15: 5 μL of Invitrogen™ BenchMark™ Protein Ladder; lanes 7, 14: 15 μL of Invitrogen™ MagicMark™ XP Western Protein Standard; lanes 8, 13: 5 μL of Invitrogen™ HiMark™ Unstained Protein Standard. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Figure 30. How to use the XCell4 SureLock Midi-Cell with 4 gels. Discover Tetra Electrophoresis Cores and precast gels bundles at thermofisher.com/tetracore 56 Tetra Electrophoresis Cores Invitrogen™ Tetra Electrophoresis Cores allow you to run highperformance Invitrogen™ precast protein gels in your Bio-Rad™ Mini-PROTEAN™ Tetra Cell tank. The Tetra Electrophoresis Cores are easy to assemble and use. The set includes: • Tetra Electrophoresis Primary Core—run up to 2 gels • Tetra Electrophoresis Companion Core—run two additional gels when combined with the primary core • Buffer dam—run 1 gel when used in combination with the primary core or 3 gels when used in combination with the primary and companion cores • Fast run times—shorten the electrophoretic run to 14 minutes with rapid protocols Different tank, same exceptional performance Invitrogen precast mini gels show excellent separation whether the electrophoretic separation is performed with the Tetra Electrophoresis Cores in the Bio-Rad Mini-PROTEAN Tetra Cell or in the Invitrogen Mini Gel Tank (Figure 32). Tetra Electrophoresis Cores allow the adoption of rapid protocols that reduce the overall separation time of Bolt Bis-Tris Plus and Novex Tris-Glycine Plus mini gels (Figure 33). Watch the Tetra Electrophoresis Cores video Figure 32. Equivalent performance of NuPAGE Bis-Tris 4–12% gels run with the Mini Gel Tank and with Tetra Electrophoresis Cores in the Bio-Rad Mini-PROTEAN Tetra Cell. Gels were loaded with Thermo Scientific™ PageRuler™ Unstained Protein Ladder and dilutions of HEK293 lysate. After electrophoresis in MES running buffer, gels were stained with SimplyBlue SafeStain. Figure 33. Similar performance between rapid and standard protocols. Novex Tris-Glycine Plus 4–20% gels were loaded with PageRuler Unstained Protein Ladder and dilutions of HEK293 lysate. After electrophoresis, gels were stained with SimplyBlue SafeStain. Mini Gel Tank Tetra Electrophoresis Primary Core in the Mini-PROTEAN Tetra Cell Standard protocol (225 V, 34 min) Rapid protocol (300 V, 20 min) 57 A B C D Learn more at thermofisher.com/tetracores 1. Insert the gel into the core with the short plate facing forward. Insert a second gel or the buffer dam on the opposite side (A). Note: remove the comb and the tape at the bottom of the gel before placing it in the core. 2. Using both hands, close the clamping frame wings onto the core to lock them in place (B). Repeat steps 1 and 2 for the companion core. 3. Insert the assembled cores into the Mini-PROTEAN Tetra Cell electrophoresis tank (C). 4. Fill the tank with 1X running buffer: 170 mL per core for the inner chamber and 550 mL or 800 mL for one or both cores, respectively, for the outer chamber. 5. Load samples, then place the lid on the tank aligning the polarity signs. Connect the cables to the power supply and start the run (D). For recommended run conditions with different gel types, download the quick reference card. Figure 34. How to use the Tetra Electrophoresis Cores with 4 gels. PowerEase Touch Power Supplies Easy touchscreen programming and operation The Invitrogen™ PowerEase™ Touch Power Supplies make setting up custom protocols or selecting one of the several preprogrammed gel electrophoresis and transfer methods a breeze with an improved 4.3-inch backlit LCD touchscreen display and user interface. The power supplies are ideal for DNA or RNA electrophoresis, SDS-PAGE, and native PAGE. The PowerEase Touch Power Supplies offer four sets of output jacks that can be used simultaneously and three modes: constant voltage, constant current, and constant power for flexibility of use and efficiency. The sturdy polyurethane feet and stackable housing design allow stacking of power supplies for a reduced footprint on the lab bench. • Ease of use—LCD touchscreen display and user interface show clear menu prompts for easy use by hand or stylus and convenient monitoring of run progress • Convenient—four sets of output terminals allow running of multiple electrophoresis units • Customizable—program up to 100 custom methods, 20 steps per method, 999 minutes per step, or select one of several preprogrammed Invitrogen gel electrophoresis and transfer methods • Safety—features automatic No Load, Over Temperature, Over Voltage, Over Current, Load Change, and Ground Leak detection Use the following table to choose the power supply that works best for your application. 58 Learn more at thermofisher.com/powerease Recommended power supplies for protein electrophoresis applications. Model Mini gel runs Mini gel transfers Midi gel runs Midi gel transfers IEF gels PowerEase Touch 120W Power Supply 4 2 2 No No PowerEase Touch 350W Power Supply 12 8 8 4 No PowerEase Touch 600W Power Supply 16 8 14 4 8 PowerEase Touch HV Power Supply 4 2 2 No 8 59 60 Mini gel running conditions in Invitrogen mini gel electrophoresis chamber systems. Gel type Voltage (V) Approximate run time (min) Bolt Bis-Tris Plus gel (MES buffer) 200 20 Bolt Bis-Tris Plus gel (MOPS buffer) 200 35 NuPAGE Bis-Tris gel (MES buffer) 200 30 NuPAGE Bis-Tris gel (MOPS buffer) 200 42 Novex Tris-Glycine gel, WedgeWell format (denatured) 225 25–40 Novex Tris-Glycine gel, WedgeWell format (native) 125 60–90 NuPAGE 3–8% Tris-Acetate gel (denatured) 150 50 NuPAGE 3–8% Tris-Acetate gel (native) 150 100 Novex 10–20% Tricine gel 125 65 NativePAGE 3–12% gel 150 80 Novex IEF gel, pH 3–10 100 60 200 60 500 30 Novex Zymogram Plus gel (gelatin) 125 90 Note: Run times may vary depending on the power supply and gel percentage. Midi gel running conditions in Invitrogen midi gel electrophoresis chamber systems. Gel type Voltage (V, constant) Estimated run time (min) Bolt Bis-Tris Plus gel (MES buffer) 200 20 Bolt Bis-Tris Plus (MOPS buffer) 200 28 NuPAGE Bis-Tris gel (MES buffer) 200 25 NuPAGE Bis-Tris gel (MOPS buffer) 200 38 Novex Tris-Glycine Plus gel (denatured) 225 33 Novex Tris-Glycine Plus gel (native) 225 90 NuPAGE Tris-Acetate gel (denatured) 150 54 NuPAGE Tris-Acetate gel (native) 150 120 Novex Tricine gel (denatured) 125 63 NativePAGE Bis-Tris gel 150 120 Note: Run times may vary depending on the power supply and gel percentage.. Running gels Run conditions for rapid protocols to achieve faster electrophoresis times. Gel type Voltage (V, constant) Estimated run time (min) Bolt Bis-Tris gel (MES buffer) 250 14 Novex Tris-Glycine gel (denatured) 300 20 Power output for rapid protocols. Number of mini gels 100 W 200 W 300 W Up to 4 ✓ ✓ ✓ Up to 8 X ✓ ✓ Up to 12 X ✓ ✓ Up to 16 X X ✓ Note: Run time may vary depending on gel percentage and the power supply used for electrophoresis. For the fastest run time, equilibrate gels to room temperature if stored at 4°C. Use caution when discarding the running buffer after the run is complete as rapid protocols will result in hotter buffer temperatures. To reduce the heat generated during the rapid run, always fill the tank to the 4-gel fill line even if running only 2 gels. 61 1. Water wash 3. Water wash DI H2O DI H2O 2. Fix F S 4. Stain 5. Destain Once protein bands have been separated by electrophoresis, they can be directly visualized using different methods of in-gel detection. Over the past several decades, demand for improved sensitivity and compatibility with downstream applications and detection instrumentation has driven the development of several basic staining methods. Each method has particular advantages and disadvantages, and a number of specific formulations for each type of method helps provide optimal performance for various situations. Typically, these stains can be classified broadly based on the molecules that help visualize the proteins: Coomassie stains • Thermo Scientific™ PageBlue™ Protein Staining Solution • Invitrogen™ SimplyBlue™ SafeStain • Thermo Scientific™ Imperial™ Protein Stain Silver stains • Thermo Scientific™ Pierce™ Silver Stain Kit • Invitrogen™ SilverXpress™ Silver Staining Kit • Thermo Scientific™ Pierce™ Silver Stain for Mass Spectrometry Staining gels Protein stains Fluorescent and specialty stains • Invitrogen™ SYPRO™ Orange, SYPRO Red, SYPRO Ruby gel stains • Thermo Scientific™ Pierce™ Reversible Protein Stain Kit for Nitrocellulose or PVDF Membranes • Invitrogen™ Pro-Q™ Emerald glycoprotein stain • Invitrogen™ Pro-Q™ Diamond phosphoprotein stain To visualize the proteins, a protein-specific, dye-binding or color-producing chemical reaction must be performed on the proteins within the gel. Depending on the particular chemistry of the stain, various steps are necessary to hold the proteins in the matrix and to facilitate the necessary chemical reaction. Most staining methods involve some version of the same general incubation steps: • A water wash to remove electrophoresis buffers from the gel matrix • An acid or alcohol wash to condition or fix the gel to limit diffusion of protein bands from the matrix • Treatment with the stain reagent to allow the reagent to diffuse into the gel and bind to (or react with) the proteins • Destaining to remove excess dye from the background gel matrix Depending on the particular staining method, two or more of these functions can be accomplished with one step. For example, a dye reagent that is formulated in an acidic buffer can effectively fix and stain in one step. Conversely, certain functions require several steps. For example, silver staining requires both a staining reagent step and a development step to produce the colored reaction product. Learn more at thermofisher.com/proteinstains 62 Coomassie dye–based protein gel stains. SimplyBlue SafeStain Imperial Protein Stain PageBlue Protein Staining Solution Type G-250 R-250 G-250 Limit of detection >7 ng 3 ng 5 ng Time to stain* 12 min 12 min 30 min Compatible with: PVDF membranes Yes Yes Yes Nitrocellulose membranes No No No Reusable No No Yes (up to 3 times) Mass spectrometry compatible Yes Yes Yes Color Purple Purple Blue-green Feature Free of methanol and acetic acid Photographs better than Coomassie G-250 dye Free of methanol and acetic acid Advantages Rapid, sensitive, completely nonhazardous staining (does not require methanol or acetic acid fixatives or destains) Fast, ultrasensitive protein detection Cost-effective option for fast, sensitive staining * Approximate staining time using microwave oven. Coomassie dye–based protein gel stains Convenient, ready-to-use reagents with no permanent chemical modification Our Coomassie stains provide sensitive protein detection along with simplified protocols. Staining protocols and example data are shown for SimplyBlue SafeStain (Figures 35, 38, 39), Imperial Protein Stain (Figures 36, 40), and PageBlue Protein Staining Solution (Figure 37). Learn more at thermofisher.com/coomassiestains The most common methods of in-gel protein detection use stains with Coomassie dye. These stains use either the G-250 (colloidal) or R-250 form of the dye. Colloidal Coomassie stain can be formulated to effectively stain proteins within one hour and require only water (no methanol or acetic acid) for destaining. Key features: • Simple—Coomassie dye–based formulations are easy to formulate and are widely used • Easy to use—simply soak the gel in staining solution, and destain to observe protein bands • Economical—Coomassie dye–based formulations are cost-effective • Flexible—useful for qualitative visualization, quantitative densitometry, and gel excision and analysis by mass spectrometry 63 Protocols 2. Add PageBlue Protein Staining Solution. S DI H2O 3. Rinse gel 2 tim es with DI water. DI H2O 4. Wash gel 1 time with DI water. 1. Wash gel 3 times with ultrapure DI water. DI H2O 30 minutes 1 hour <1 minute 5 minutes Figure 37. PageBlue Protein Staining Solution protocol. DI H2O 3. Wash gel with 100 mL of DI water. DI H2O 4. Additional water wash with 100 mL of DI water for increased sensitivity. 1. Wash gel 3 times with ultrapure DI water. DI H2O 2. Add SimplyBlue SafeStain. S 5 minutes 1 hour 1 hour 1 hour Figure 35. SimplyBlue SafeStain protocol. Figure 36. Imperial Protein Stain protocol. 2. Add Imperial Protein Stain. S DI H2O 1. Wash gel 3 times 3. Water destain. with DI water. DI H2O 15 minutes 5 minutes–1 hour 15 minutes–overnight 64 Example data Figure 40. Enhanced sensitivity and clear background using Imperial Protein Stain. For even greater sensitivity and reduced background, gels can be stained with Imperial Protein Stain for 1 hour and washed with water from 1 hour to overnight. Lane 1: BSA only (6 μg); lanes 2–9: loaded with 1,000, 200, 100, 50, 25, 12, 6, and 3 ng protein. Figure 38. Sensitive staining results with SimplyBlue SafeStain. The following samples were separated on an Invitrogen™ NuPAGE™ 4–12% Bis-Tris gel and then stained with SimplyBlue SafeStain. Lane 1: 6 μg protein mix; lane 2: 1 μg rabbit IgG; lane 3: 1 μg reduced BSA; lane 4: 5 μg E. coli lysate; lane 5: 20 ng reduced BSA; lane 6: 10 ng reduced BSA; lane 7: 7 ng reduced BSA; lane 8: 3 ng reduced BSA; lane 9: 10 μL Invitrogen™ Mark12™ Unstained Standard (blend of 12 purified proteins); lane 10: 5 μL Mark12 Unstained Standard. 5-minute stain; 15-minute water destain 1-hour stain; 2-hour water destain 1-hour stain; overnight water destain Rabbit IgG BSA Protein A Protein G Lysozyme 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Figure 39. Two-dimensional electrophoresis analysis of spinach chloroplast extract; staining with SimplyBlue SafeStain. A spinach chloroplast extract was prefractionated in the Invitrogen™ ZOOM™ IEF Fractionator, and the individual fractions were then separated by 2D electrophoresis using narrow pH–range Invitrogen™ ZOOM™ IPG strips and an Invitrogen™ NuPAGE™ 4–12% Bis-Tris ZOOM™ gel. Gels were stained using SimplyBlue SafeStain. 1 2 3 4 5 6 7 8 9 10 ? Did you know Staining with a Coomassie stain prior to silver staining allows for more uniform staining of certain proteins since silver ions can interact with certain functional groups such as carboxylic acid groups, imidazole, sulfhydryls, and amines. 65 Silver staining is the most sensitive colorimetric method for detecting total protein, and functions by the deposition of metallic silver at the location of protein bands. Silver ions (from silver nitrate in the stain reagent) interact and bind with certain protein functional groups. The strongest interactions occur with carboxylic acid groups (Asp and Glu), imidazole (His), sulfhydryl groups (Cys), and amines (Lys). Various sensitizer and enhancer reagents are important for controlling the specificity and efficiency of silver ion binding to proteins and effective conversion (development) of the bound silver to metallic silver. Silver stains Ultrasensitive stains with optimized protocols and manufactured for minimal variability We offer highly sensitive silver stains with short protocol times that are also compatible with mass spectrometry. The Invitrogen™ SilverXpress™ Silver Staining Kit provides nanogram-level sensitivity with minimal background (Figures 41, 42), while the Thermo Scientific™ Pierce™ Silver Stain Kit provides protocol flexibility (Figures 43, 44). Learn more at thermofisher.com/silverstains Key features: • Sensitive—silver stains are highly sensitive stains that allow for visualization of proteins at subnanogram levels • Easy to use and flexible—silver stains are optimized for minimal steps and have the flexibility to accommodate shorter or longer protocols • Workflow compatible—our mild chemical formulations help ensure compatibility with mass spectrometry and sequencing • Robust performance—detailed protocol enables consistent results with clear background 66 Silver stain kits. Pierce Silver Stain for Mass Spectrometry Pierce Silver Stain Kit SilverXpress Silver Staining Kit Components (steps) 6 (7) 4 (7) 5 (9) Time required 1 hr 13 min 1 hr 30 min 2 hr Limit of detection 0.25 ng 0.25 ng 0.86 ng Mass spectrometry compatible Yes Yes Yes Storage Room temperature Room temperature 4°C Stability 1 year 1 year 6 months Advantages • Fast and sensitive staining and destaining of protein gels • Optimized for peptide recovery after in-gel trypsin digestion for mass spectrometry • Flexible gel fixation (15–30 min to overnight) and staining (1–30 min) • Rapid, ultrasensitive, and versatile silver stain system • Flexible gel fixation (30 min–overnight) and staining (5 min–20 hr) • Nanogram-level sensitivity for silver staining with minimal background Figure 41. SilverXpress Silver Staining Kit protocol. Figure 42. Crystal clear background with the SilverXpress Silver Staining Kit. Samples were separated on a NuPAGE 4–12% Bis-Tris gel and stained using the SilverXpress kit. Lanes 1, 10: Mark12 Unstained Standard (blend of 12 purified proteins) diluted 1:4; lane 2: Mark12 Unstained Standard diluted 1:8; lane 3: Mark12 Unstained Standard diluted 1:16; lane 4: Mark12 Unstained Standard diluted 1:32; lane 5: Mark12 Unstained Standard diluted 1:64; lane 6: 1.6 ng BSA; lane 7: 0.8 ng BSA; lane 8: E. coli lysate diluted 1:20; lane 9: E. coli lysate diluted 1:80. 1 2 3 4 5 6 7 8 9 10 Protocols and example data 1. Wash gel with water. H2O 9. Decant Stopping Solution and wash gel 3 times with ultrapure water. H2O H2O 2. Fix gel in Fixing Solution for 10 minutes. F 4. Decant Sensitizing Solution and rinse gel 2 times with ultrapure water. H2O 6. Decant Staining Solution and rinse gel 2 times with ultrapure water. SZ 3. Decant Fixing Solution and incubate gel in 2 changes of Sensitizing Solution. SS 8. Add Stopping Solution directly to gel when desired staining intensity is reached. 5. Incubate gel in Staining Solution. S S 7. Incubate gel in Developing Solution. D D 1. Wash gel with water. H2O 9. Decant Stopping Solution and wash gel 3 times with ultrapure water. H2O H2O 2. Fix gel in Fixing Solution for 10 minutes. F 4. Decant Sensitizing Solution and rinse gel 2 times with ultrapure water. H2O 6. Decant Staining Solution and rinse gel 2 times with ultrapure water. SZ 3. Decant Fixing Solution and incubate gel in 2 changes of Sensitizing Solution. SS 8. Add Stopping Solution directly to gel when desired staining intensity is reached. 5. Incubate gel in Staining Solution. S S 7. Incubate gel in Developing Solution. D D 1. Wash gel with water. H2O 9. Decant Stopping Solution and wash gel 3 times with ultrapure water. H2O H2O 2. Fix gel in Fixing Solution for 10 minutes. F 4. Decant Sensitizing Solution and rinse gel 2 times with ultrapure water. H2O 6. Decant Staining Solution and rinse gel 2 times with ultrapure water. SZ 3. Decant Fixing Solution and incubate gel in 2 changes of Sensitizing Solution. SS 8. Add Stopping Solution directly to gel when desired staining intensity is reached. 5. Incubate gel in Staining Solution. S S 7. Incubate gel in Developing Solution. D D 1. Wash gel with water. H2O 9. Decant Stopping Solution and wash gel 3 times with ultrapure water. H2O H2O 2. Fix gel in Fixing Solution for 10 minutes. F 4. Decant Sensitizing Solution and rinse gel 2 times with ultrapure water. H2O 6. Decant Staining Solution and rinse gel 2 times with ultrapure water. SZ 3. Decant Fixing Solution and incubate gel in 2 changes of Sensitizing Solution. SS 8. Add Stopping Solution directly to gel when desired staining intensity is reached. 5. Incubate gel in Staining Solution. S S 7. Incubate gel in Developing Solution. D D 67 Figure 43. Pierce Silver Stain Kit protocol. Lysate staining 2 minutes, 30 seconds Development time 2 minutes, 30 seconds MW marker staining Figure 44. The Pierce Silver Stain Kit exhibits excellent sensitivity. In standard mini gels, proteins are detectable at greater than 0.25 ng per band or spot. 1. Wash 2 x 5 minutes with ultrapure water. H2O 2. Fix 2 x 15 minutes in EtOH/acetic acid. EtOH Acetic acid F F 4. Mix Sensitizer. Sensitize for 1 minute. Wash 2 x 1 minute. SZ SZ 3. Incubate 2 x 5 minutes with 10% EtOH. Wash. 10% EtOH 5. Mix Silver Stain. Stain for 5 minutes. Wash 2 x 20 seconds. S S 6. Mix Developer. Develop for 2–3 minutes. D D 7. Remove Developer. Stop with 5% acetic acid for 10 minutes. 5% acetic acid 1. Wash 2 x 5 minutes with ultrapure water. H2O 2. Fix 2 x 15 minutes in EtOH/acetic acid. EtOH Acetic acid F F 4. Mix Sensitizer. Sensitize for 1 minute. Wash 2 x 1 minute. SZ SZ 3. Incubate 2 x 5 minutes with 10% EtOH. Wash. 10% EtOH 5. Mix Silver Stain. Stain for 5 minutes. Wash 2 x 20 seconds. S S 6. Mix Developer. Develop for 2–3 minutes. D D 7. Remove Developer. Stop with 5% acetic acid for 10 minutes. 5% acetic acid 1. Wash 2 x 5 minutes with ultrapure water. H2O 2. Fix 2 x 15 minutes in EtOH/acetic acid. EtOH Acetic acid F F 4. Mix Sensitizer. Sensitize for 1 minute. Wash 2 x 1 minute. SZ SZ 3. Incubate 2 x 5 minutes with 10% EtOH. Wash. 10% EtOH 5. Mix Silver Stain. Stain for 5 minutes. Wash 2 x 20 seconds. S S 6. Mix Developer. Develop for 2–3 minutes. D D 7. Remove Developer. Stop with 5% acetic acid for 10 minutes. 5% acetic acid 1. Wash 2 x 5 minutes with ultrapure water. H2O 2. Fix 2 x 15 minutes in EtOH/acetic acid. F F 4. Mix Sensitizer. Sensitize for 1 minute. Wash 2 x 1 minute. SZ SZ 3. Incubate 2 x 5 minutes with 10% EtOH. Wash. 10% EtOH 5. Mix Silver Stain. Stain for 5 minutes. Wash 2 x 20 seconds. S S 6. Mix Developer. Develop for 2–3 minutes. D D 7. Remove Developer. Stop with 5% acetic acid for 10 minutes. 5% acetic acid 68 Fluorescent gel stains are designed for use in 1D and 2D PAGE and offer sensitivities similar to those of silver staining techniques. Invitrogen™ SYPRO™ protein stains are easy-to-use fluorescent stains for the detection of proteins separated by PAGE. Stained proteins can be viewed with a standard UV or blue-light transilluminator or with a laser scanner. Fluorescent protein gel stains Rapid, highly sensitive fluorescent stains for total protein detection after electrophoresis SYPRO protein stains. SYPRO Ruby stain SYPRO Orange stain SYPRO Red stain Limit of detection 0.25 ng 4–8 ng 4–8 ng Stain and destain time 90 min microwave; 18 hr standard ~1 hr ~1 hr Ex/Em 280 nm, 450/610 nm 300 nm, 470/510 nm 300 nm, 550/630 nm Ease of use Ready to use Supplied as stock solution Supplied as stock solution Compatible applications Mass spectrometry, IEF, 2D gels, on-membrane staining Mass spectrometry, IEF, 2D gels, on-membrane staining Mass spectrometry, IEF, 2D gels, on-membrane staining Our specialty protein stains include in-gel phosphoprotein and glycoprotein detection staining kits. Specialty protein stains Specialty protein stains. Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain Kit Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain Kit Pro-Q Diamond Phosphoprotein Gel Staining Kit Detects Glycoproteins Glycoproteins Phosphoproteins Sensitivity 4 ng glycoprotein per band 0.5 ng glycoprotein per band 1–16 ng phosphoprotein per band Stain and destain time ~6 hr ~5 hr 4–5 hr Ex/Em 510/520 nm 280/530 nm 555/580 nm Advantages Selective staining of glycoproteins Selective staining of glycoproteins Selective staining of phosphoproteins Learn more at thermofisher.com/specialtystains Learn more at thermofisher.com/fluorescentstains Features: • Simple—no destaining or timed steps required; minimal hands-on time • Quantitative—linear quantitation range over two orders of magnitude with low protein-to-protein variability • Highly sensitive—typically more sensitive than Coomassie dye–based stains and equivalent to silver stains 69 The Invitrogen™ No-Stain™ Protein Labeling Reagent offers a flexible, accurate, rapid, and reliable method to visualize and normalize proteins in a gel or on a membrane (post-transfer). It forms covalent bonds with lysine residues in proteins in gels or on membranes within 10 minutes without the need for destaining. Increasing incubation time or doubling the reagent concentration produces a proportionally stronger signal for improved detection of low-abundance proteins. The results can be instantly visualized using any commonly available imager, with nanogram-level sensitivity (Figure 45). No-Stain Labeling Reagent Instant visualization of proteins in gels Figure 45. Quantitative gel staining with No-Stain Protein Labeling Reagent. A Bolt 4–12% Bis-Tris Plus gel was loaded with HeLa cell lysate containing 2.5 to 80 μg of protein, and electrophoresed with MES running buffer. After electrophoresis, the proteins were labeled in-gel with No-Stain Protein Labeling Reagent, and the gel was imaged using an Invitrogen™ iBright™ imager with the transilluminator for excitation (490–520 nm) and the 565–615 nm emission filter. For details on total protein normalization for quantitative western blots using No-Stain Labeling Reagent, go to thermofisher.com/nostain Features: • Versatile—compatible with all gel chemistries and with downstream gel staining, protein transfer, immunoblotting, and mass spectrometry analysis • Flexible visualization—use a wide range of excitation sources, including UV and green fluorescence light (optimal imaging conditions: Ex. max: ~488 nm, Em. max: 590 nm) • Broad linear dynamic range—1–80 μg total protein loaded per well • Sensitive—protein bands are detected down to 20 ng, and the signal is compatible with downstream antibody detection No-Stain protocol for labeling protein on gels or transferred blots * For Tris-glycine gels and membranes derived from Tris-glycine gels, washing for two minutes with ultrapure water, 4 and 2 times, respectively, is necessary to remove glycine before performing labeling with the No-Stain Protein Labeling Reagent. Key features • Automated tray recognition—the iGlow system identifies the tray and activates only the matching light source • Programmable imaging methods—save capture settings for automated routine imaging • Onboard image editing—make quick adjustments right after capture, and export or print your updated image • Safe gel excision—blue LED light provides a safer alternative than UV band excision • Quick and simple installation—easy to set up and get started 70 Gel imaging and documentation Simply brilliant one-touch gel imaging Capture, analyze, and share images of your protein and nucleic acid gels with ease. The Invitrogen™ iGlow™ Gel Documentation System supports one-touch gel imaging with an intuitive touchscreen interface and free, integrated software—making it quick and easy to visualize, enhance, and export high-quality images. Featuring a high-resolution camera and an advanced LED light source, the iGlow system is compatible with the most common polyacrylamide and agarose gel stains. The versatile imaging toolkit is composed of UV, blue, and white light trays, along with a safety guard screen designed for safe and efficient excision of DNA bands. Discover how the iGlow Gel Documentation System simplifies everyday gel imaging in our video overview. 71 Example protein and DNA gel images captured with the iGlow Gel Documentation System Gel stains compatible with the iGlow system Application Gel stain Excitation source UV Blue White DNA Ethidium bromide ✓ Invitrogen™ SYBR™ Safe ✓ Invitrogen™ SYBR™ Gold ✓ Invitrogen™ SYBR™ Green ✓ Biotium™ GelRed™ ✓ Biotium™ GelGreen™ ✓ Midori Green ✓ Protein Invitrogen™ SYPRO™ Ruby ✓ Invitrogen™ SYPRO™ Orange ✓ Invitrogen™ SYPRO™ Red ✓ Invitrogen™ SYPRO™ Tangerine ✓ Invitrogen™ Pro-Q™ Diamond Phosphoprotein ✓ Invitrogen™ Coomassie Fluor™ Orange ✓ Bio-Rad™ Oriole™ ✓ Bio-Rad™ Flamingo™ ✓ Invitrogen™ SimplyBlue™ SafeStain ✓ Thermo Scientific™ Pierce™ Silver Stain ✓ * Most blue-light excitable dyes can also be visualized under UV illumination, and many UV-excitable dyes are compatible with blue-light imaging, though signal intensity may vary. Learn more about the iGlow Gel Documentation System at thermofisher.com/iglow SDS-PAGE gel, Invitrogen™ SimplyBlue™ SafeStain SDS-PAGE gel, Thermo Scientific™ Pierce™ Silver Stain Kit Agarose gel, Invitrogen™ UltraPure™ Ethidium Bromide SDS-PAGE gel, Invitrogen™ SYPRO™ Red Protein Gel Stain 72 Key products for western blot transfer: Wet Semi-dry Dry Mini Blot Module Invitrogen™ Power Blotter Invitrogen™ iBlot™ 3 Dry Blotting System Key products for western blot detection: Automated detection Invitrogen™ iBind™ Flex Western Device Invitrogen™ Bandmate™ Automated Western Blot Processor Invitrogen™ iBright™ Imaging Systems Manual detection Blocking buffers Wash buffers Detergents Enhancers Substrates Stripping buffers X-ray film Learn more at thermofisher.com/western Western blotting 73 Appendix Protocol quick references To learn more about our various gel products and their uses, check out these links to protocols that describe how to use the products. • Bolt Bis-Tris Plus mini gels • NuPAGE Bis-Tris mini gels • NuPAGE Bis-Tris midi gels • NuPAGE Tris-Acetate mini gels • NuPAGE Tris-Acetate midi gels • NativePAGE Bis-Tris gels • Novex Tricine gels • Novex IEF gels • Zymogram Plus gels 74 Troubleshooting tips Protein gel electrophoresis Gel electrophoresis troubleshooting Observation Possible cause Suggested solution Protein bands lose resolution, lanes have streaks and are not straight Too much protein loaded per lane Reduce the sample loads. The maximum recommended sample load for optimal resolution in mini gels with 10, 12, 15, or 17 wells is 0.5 μg per band or about 10–15 μg of cell lysate per lane. Viscous samples, streaks at sample lane edges, dumbbell-shaped bands, lane widening Excess salt (ammonium sulfate) in sample during gel electrophoresis Perform dialysis to decrease salt concentration. Use a small dialysis device such as the Thermo Scientific™ Slide-A-Lyzer™ MINI Dialysis Device, 0.5 mL (Cat. No. 88401). Concentrate and resuspend samples in lower-salt buffer prior to electrophoresis. Use small-volume concentrators such as Thermo Scientific™ Pierce™ Protein Concentrators PES, 0.5 mL (Cat. No. 88513). Make sure that the salt concentration does not exceed 100 mM. Protein aggregation resulting in narrow lanes that cannot be interpreted DNA contamination—genomic DNA in the cell lysate may cause the sample to become viscous, resulting in protein aggregation, which can affect protein migration patterns and resolution Shear genomic DNA to reduce viscosity before loading the sample. Uneven sample lanes, lane widening Excess salt (sodium chloride) in sample during gel electrophoresis. High salt concentrations result in increased conductivity, which affects protein migration and can result in protein bands spreading into adjacent lanes containing samples with normal salt concentrations Perform dialysis to decrease salt concentration. Use a small dialysis device such as the Slide-A-Lyzer MINI Dialysis Device, 0.5 mL (Cat. No. 88401). Concentrate and resuspend samples in lower-salt buffer prior to electrophoresis. Use small-volume concentrators such as Pierce Protein Concentrators PES, 0.5 mL (Cat. No. 88513). Make sure that the salt concentration does not exceed 100 mM. High detergent concentration (e.g., SDS or Triton X-100 detergent) in gel electrophoresis. Detergents form mixed micelles with the anionic detergent SDS in the gel and migrate down into the gel; they interfere with the SDS–protein binding equilibrium Most of the nonionic detergents (e.g., Triton X-100, NP-40, and Tween 20 detergents) interfere with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Keep the ratio of SDS to nonionic detergent at 10:1 or greater to minimize these effects. Use detergent removal columns or the Thermo Scientific™ Pierce™ SDS-PAGE Sample Prep Kit (Cat. No. 89888) to remove excess detergent. High concentration of RIPA (radioimmunoprecipitation assay) buffer results in widening of lanes and significant streaking during electrophoresis Dilute samples before electrophoresis to lower the final concentration of lysis buffer to prevent buffer-related defects. Shadow at lane edges Excess reducing agent in the lysis or sample buffer The final concentration of reducing agents for SDS-PAGE should be less than 50 mM for DTT (dithiothreitol) and TCEP (tris(2-carboxyethyl) phosphine), and less than 2.5% for β-ME (β-mercaptoethanol). 75 Gel electrophoresis troubleshooting (cont.) Observation Possible cause Suggested solution Run taking longer time with recommended voltage Running buffer too dilute Make fresh running buffer and use a 1X dilution. Current too high and excessive heat generated with recommended voltage Running buffer too concentrated Make fresh running buffer and use a 1X dilution. Current too low or no current with recommended voltage Incomplete circuit Remove the tape from the bottom of the gel cassette prior to electrophoresis. Make sure the buffer covers sample wells; check the wire connections on the buffer core. Streaking of proteins Sample overload Load less protein. High salt concentration in sample Decrease the sample salt concentration by dialysis or gel filtration. Sample precipitates Increase the concentration of SDS in the sample. Contaminants such as lipids or DNA complexes in sample Centrifuge or clarify the sample to remove particulate contaminants. Treat sample with nuclease(s). Poorly poured gel Make sure the gel is poured evenly and all at once. Fuzzy bands Protein sample only partially denatured Fully denature the protein. Protein sample only partially reduced Make sure a sufficient amount of DTT or β-mercaptoethanol is added. Gel runs for too long Watch the dye front as an indicator for proper running time. Dumbbell-shaped bands or “smiling” bands Loading a large volume of sample causes incomplete stacking Load appropriate volume of sample. If the sample is too dilute, concentrate it using ultrafiltration. Uneven electric field during run Try to make sure the loading is symmetrical if the protein concentration is known. Uneven surface of the resolving gel Try to make the resolving gel surface even while pouring the gel. Expired gels Use the gels before the specified expiration date. Note: NuPAGE gels have an extended 12-month shelf life, minimizing the risk of having expired gels. 76 Protein gel electrophoresis chamber systems Mini Gel Tank troubleshooting Observation Possible cause Suggested solution Run taking longer than usual Buffers are too dilute Check buffer recipe; dilute from concentrate or remake if necessary. Buffer chamber is leaking Make sure the cassette clamp is firmly seated, the gaskets are in place, and the cassette clamp is locked. Current is set too low Set correct current. Current reading on power supply is zero or very low Tape left on the bottom of the cassette Remove tape from bottom of cassette. Connection to power supply not complete Check all connections with a voltmeter for conductance. Insufficient buffer level Make sure there is sufficient buffer in the electrophoresis tank to cover the wells of the gel. Run is faster than normal with poor resolution Buffers are too concentrated or incorrect Check buffer recipe; dilute or remake if necessary. Current is set at a higher limit Decrease current to recommended running conditions (see page 60). Cannot see the sample wells to load sample There is little contrast between the sample well and the rest of the gel Mark cassette at the bottom of the wells with a marker pen prior to placing the cassette in the electrophoresis tank. XCell SureLock Mini-Cell troubleshooting Observation Possible cause Suggested solution Run taking longer than usual Buffers are too dilute Check if buffer was diluted properly. Check buffer recipe; dilute from concentrate or remake if necessary. Upper buffer chamber is leaking Make sure the buffer core is firmly seated, the gaskets are in place, and the gel tension lever is locked. Voltage is set too low Set correct voltage. Current reading on power supply is zero or very low Tape left on the bottom of the cassette Remove tape from bottom of cassette. Connection to power supply not complete Check all connections with a voltmeter for conductance. Insufficient buffer level Make sure the upper buffer (cathode) is covering the wells of the gel. Be sure there is sufficient buffer in the lower buffer chamber to cover the slot at the bottom of the gel. Run is faster than normal with poor resolution Buffers are too concentrated or incorrect Check buffer recipe; dilute or remake if necessary. Voltage, current, or wattage is set at a higher limit Decrease power conditions to recommended running conditions (see page 60). Cannot see the sample wells to load sample There is little contrast between the sample well and the rest of the gel Mark cassette at the bottom of the wells with a marker pen prior to assembling the upper buffer chamber. Illuminate the bench area with a light source placed directly behind the XCell SureLock unit. 77 SureLock Tandem Midi Gel Tank troubleshooting Observation Possible cause Suggested solution Run takes longer than normal Buffers were too dilute Check buffer recipes. Do not reuse buffers. Remake buffer if necessary. Buffer chamber was leaking Make sure the cassette clamp is firmly seated, the gaskets are in place, and the cassette clamps are locked. Voltage and/or current was set too low Set the correct voltage and/or current. See “Running conditions” in the “Gel electrophoresis protocol” (p. 10) of the user guide for more details. Run is faster than normal, with poor resolution Incorrect running buffer was used or buffer was too concentrated Check buffer recipe. Dilute or remake buffer if necessary. Voltage and/or current was set too high Decrease voltage and/or current to recommended running conditions. See “Running conditions” in the “Gel electrophoresis protocol” (p. 10) of the user guide for more details. Current reading on power supply is zero or very low Tape was left on the bottom of the cassette Remove the tape from the cassette. Connection to power supply was not complete Check conductance on all connections using a voltmeter. Note: Do not check connections if not trained in this procedure. Contact Technical Support for further help. Insufficient buffer level Make sure there is sufficient buffer in the tank. The cathode (inside) chamber needs to be filled to above the wells (~170 mL), and the anode (outside) chamber needs to be filled to the red fill line (~350 mL). See “Fill tank with running buffer and load samples” in the “Gel electrophoresis protocol” (p. 12) of the user guide for more details. Cannot see the sample wells to load the sample Difficult to see contrast between the sample well and the rest of the gel Mark cassette at the bottom of the wells with a marker pen prior to placing the cassette in the electrophoresis tank. Current reading on power supply is much higher than expected, or maxed out Too many gels were being run at once using one power supply Check the power limits of the power supply being used and use additional power supplies if needed. Cassette was not properly clamped Ensure that the cassette clamp is firmly seated, the gaskets are in place, and the cassette clamps are in the locked position. See “Insert cassette” in the “Gel electrophoresis protocol” (p. 11) of the user guide for details. Tank was overfilled with running buffer If the buffer level is above the height of the gel, the cathode and anode chambers are in direct contact, resulting in an electrical short circuit. Ensure correct buffer volumes. The cathode buffer level should be covering the wells but not above the height of the gel (~170 mL). The anode buffer level should be filled to the red fill line marked on the outside of the tank (~350 mL). See “Fill tank with running buffer and load samples” in the “Gel electrophoresis protocol” (p. 12) of the user guide for more details. Proteins are not migrating into the gel The cassette is installed backwards in the tank Install the cassette in the correct orientation, with the well opening facing toward the cathode (center of the tank). The power cable is installed backwards into the power supply Install the power cable in the correct orientation (red to red, black to black). 78 Ordering information Product Quantity Cat. No. Gel welcome packs Novex WedgeWell Welcome Pack, 6%, 10-well 1 kit XP0006A Novex WedgeWell Welcome Pack, 6%, 12-well 1 kit XP0006B Novex WedgeWell Welcome Pack, 6%, 15-well 1 kit XP0006C Novex WedgeWell Welcome Pack, 8%, 10-well 1 kit XP0008A Novex WedgeWell Welcome Pack, 8%, 12-well 1 kit XP0008B Novex WedgeWell Welcome Pack, 8%, 15-well 1 kit XP0008C Novex WedgeWell Welcome Pack, 10%, 10-well 1 kit XP0010A Novex WedgeWell Welcome Pack, 10%, 12-well 1 kit XP0010B Novex WedgeWell Welcome Pack, 10%, 15-well 1 kit XP0010C Novex WedgeWell Welcome Pack, 12%, 10-well 1 kit XP0012A Novex WedgeWell Welcome Pack, 12%, 12-well 1 kit XP0012B Novex WedgeWell Welcome Pack, 12%, 15-well 1 kit XP0012C Novex WedgeWell Welcome Pack, 14%, 10-well 1 kit XP0014A Novex WedgeWell Welcome Pack, 14%, 12-well 1 kit XP0014B Novex WedgeWell Welcome Pack, 14%, 15-well 1 kit XP0014C Novex WedgeWell Welcome Pack, 16%, 10-well 1 kit XP0016A Novex WedgeWell Welcome Pack, 16%, 12-well 1 kit XP0016B Novex WedgeWell Welcome Pack, 16%, 15-well 1 kit XP0016C Novex WedgeWell Welcome Pack, 4−12%, 10-well 1 kit XP0412A Novex WedgeWell Welcome Pack, 4−12%, 12-well 1 kit XP0412B Novex WedgeWell Welcome Pack, 4−12%, 15-well 1 kit XP0412C Novex WedgeWell Welcome Pack, 4−20%, 10-well 1 kit XP0420A Novex WedgeWell Welcome Pack, 4−20%, 12-well 1 kit XP0420B Novex WedgeWell Welcome Pack, 4−20%, 15-well 1 kit XP0420C Novex WedgeWell Welcome Pack, 8−16%, 10-well 1 kit XP0816A Novex WedgeWell Welcome Pack, 8−16%, 12-well 1 kit XP0816B Novex WedgeWell Welcome Pack, 8−16%, 15-well 1 kit XP0816C Novex WedgeWell Welcome Pack, 10−20%, 10-well 1 kit XP1020A Novex WedgeWell Welcome Pack, 10−20%, 12-well 1 kit XP1020B Novex WedgeWell Welcome Pack, 10−20%, 15-well 1 kit XP1020C Bolt Welcome Pack A (4−12%, 10-well) 1 kit NW0412A Bolt Welcome Pack B (4−12%, 15-well) 1 kit NW0412B Bolt Welcome Pack, 4−12%, 12-well 1 kit NW0412C Bolt Welcome Pack, 10%, 10-well 1 kit NW0010A Bolt Welcome Pack, 10%, 12-well 1 kit NW0010B Bolt Welcome Pack, 10%, 15-well 1 kit NW0010C Bolt Welcome Pack, 12%, 10-well 1 kit NW0012A Bolt Welcome Pack, 12%, 12-well 1 kit NW0012B Bolt Welcome Pack, 12%, 15-well 1 kit NW0012C Bolt Welcome Pack, 8%, 10-well 1 kit NW0008A Bolt Welcome Pack, 8%, 12-well 1 kit NW0008B Bolt Welcome Pack, 8%, 15-well 1 kit NW0008C Product Quantity Cat. No. Gel welcome packs (cont.) NuPAGE Tris-Acetate Welcome Pack, 3−8%, 10-well 1 kit EA0375A NuPAGE Tris-Acetate Welcome Pack, 3−8%, 12-well 1 kit EA0375B NuPAGE Tris-Acetate Welcome Pack, 3−8%, 15-well 1 kit EA0375C NuPAGE Tris-Acetate Welcome Pack, 7%, 10-well 1 kit EA0355A NuPAGE Tris-Acetate Welcome Pack, 7%, 12-well 1 kit EA0355B NuPAGE Tris-Acetate Welcome Pack, 7%, 15-well 1 kit EA0355C NuPAGE Bis-Tris Welcome Pack, 10%, 10-well 1 kit NP030A NuPAGE Bis-Tris Welcome Pack, 10%, 12-well 1 kit NP030B NuPAGE Bis-Tris Welcome Pack, 10%, 15-well 1 kit NP030C NuPAGE Bis-Tris Welcome Pack, 12%, 10-well 1 kit NP034A NuPAGE Bis-Tris Welcome Pack, 12%, 12-well 1 kit NP034B NuPAGE Bis-Tris Welcome Pack, 12%, 15-well 1 kit NP034C NuPAGE Bis-Tris Welcome Pack, 4−12%, 10-well 1 kit NP032A NuPAGE Bis-Tris Welcome Pack, 4−12%, 12-well 1 kit NP032B NuPAGE Bis-Tris Welcome Pack, 4−12%, 15-well 1 kit NP032C Novex Tricine Welcome Pack, 10%, 10-well 1 kit EC6675A Novex Tricine Welcome Pack, 10%, 12-well 1 kit EC6675B Novex Tricine Welcome Pack, 16%, 10-well 1 kit EC6695A Novex Tricine Welcome Pack, 16%, 12-well 1 kit EC6695B Novex Tricine Welcome Pack, 16%, 15-well 1 kit EC6695C Novex Tricine Welcome Pack, 10−20%, 10-well 1 kit EC6625A Novex Tricine Welcome Pack, 10−20%, 12-well 1 kit EC6625B Novex Tricine Welcome Pack, 10−20%, 15-well 1 kit EC6625C Learn more at thermofisher.com/proteingelwelcome 79 Product Quantity Cat. No. Bolt Bis-Tris Plus gels (8 x 8 cm) Bolt 8% Bis-Tris Plus Gels, 10-well 10 gels NW00080BOX Bolt 8% Bis-Tris Plus Gels, 12-well 10 gels NW00082BOX Bolt 8% Bis-Tris Plus Gels, 15-well 10 gels NW00085BOX Bolt 8% Bis-Tris Plus Gels, 17-well 10 gels NW00087BOX Bolt 10% Bis-Tris Plus Gels, 10-well 10 gels NW00100BOX Bolt 10% Bis-Tris Plus Gels, 12-well 10 gels NW00102BOX Bolt 10% Bis-Tris Plus Gels, 15-well 10 gels NW00105BOX Bolt 10% Bis-Tris Plus Gels, 17-well 10 gels NW00107BOX Bolt 12% Bis-Tris Plus Gels, 10-well 10 gels NW00120BOX Bolt 12% Bis-Tris Plus Gels, 12-well 10 gels NW00122BOX Bolt 12% Bis-Tris Plus Gels, 15-well 10 gels NW00125BOX Bolt 12% Bis-Tris Plus Gels, 17-well 10 gels NW00127BOX Bolt 4–12% Bis-Tris Plus Gels, 10-well 10 gels NW04120BOX Bolt 4–12% Bis-Tris Plus Gels, 12-well 10 gels NW04122BOX Bolt 4–12% Bis-Tris Plus Gels, 15-well 10 gels NW04125BOX Bolt 4–12% Bis-Tris Plus Gels, 17-well 10 gels NW04127BOX Bolt Empty Mini Gel Cassettes 20 cassettes NW2010 Bolt Empty Mini Gel Cassette Combs, 10-well 20 combs NW3010 Bolt Empty Mini Gel Cassette Combs, 12-well 20 combs NW3012 Product Quantity Cat. No. NuPAGE Bis-Tris mini gels (8 x 8 cm) NuPAGE 10% Bis-Tris Protein Gels, 1.0 mm, 1-well 10 gels NP0304BOX NuPAGE 10% Bis-Tris Protein Gels, 1.0 mm, 10-well 10 gels NP0301BOX NuPAGE 10% Bis-Tris Protein Gels, 1.0 mm, 10-well 2 gels NP0301PK2 NuPAGE 10% Bis-Tris Protein Gels, 1.0 mm, 12-well 10 gels NP0302BOX NuPAGE 10% Bis-Tris Protein Gels, 1.0 mm, 12-well 2 gels NP0302PK2 NuPAGE 10% Bis-Tris Protein Gels, 1.0 mm, 15-well 10 gels NP0303BOX NuPAGE 10% Bis-Tris Protein Gels, 1.0 mm, 9-well 10 gels NP0307BOX NuPAGE 10% Bis-Tris Protein Gels, 1.5 mm, 10-well 10 gels NP0315BOX NuPAGE 10% Bis-Tris Protein Gels, 1.5 mm, 15-well 10 gels NP0316BOX NuPAGE 12% Bis-Tris Protein Gels, 1.0 mm, 1-well 10 gels NP0344BOX NuPAGE 12% Bis-Tris Protein Gels, 1.0 mm, 10-well 10 gels NP0341BOX NuPAGE 12% Bis-Tris Protein Gels, 1.0 mm, 10-well 2 gels NP0341PK2 NuPAGE 12% Bis-Tris Protein Gels, 1.0 mm, 12-well 10 gels NP0342BOX NuPAGE 12% Bis-Tris Protein Gels, 1.0 mm, 12-well 2 gels NP0342PK2 NuPAGE 12% Bis-Tris Protein Gels, 1.0 mm, 15-well 10 gels NP0343BOX NuPAGE 12% Bis-Tris Protein Gels, 1.0 mm, 17-well 10 gels NP0349BOX NuPAGE 4–12% Bis-Tris Protein Gels, 1.0 mm, 1-well 10 gels NP0324BOX NuPAGE 4–12% Bis-Tris Protein Gels, 1.0 mm, 10-well 10 gels NP0321BOX NuPAGE 4–12% Bis-Tris Protein Gels, 1.0 mm, 10-well 2 gels NP0321PK2 NuPAGE 4–12% Bis-Tris Protein Gels, 1.0 mm, 12-well 10 gels NP0322BOX NuPAGE 4–12% Bis-Tris Protein Gels, 1.0 mm, 12-well 2 gels NP0322PK2 NuPAGE 4–12% Bis-Tris Protein Gels, 1.0 mm, 15-well 10 gels NP0323BOX NuPAGE 4–12% Bis-Tris Protein Gels, 1.0 mm, 15-well 2 gels NP0323PK2 NuPAGE 4–12% Bis-Tris Protein Gels, 1.0 mm, 17-well 10 gels NP0329BOX NuPAGE 4–12% Bis-Tris Protein Gels, 1.0 mm, 17-well 2 gels NP0329PK2 NuPAGE 4–12% Bis-Tris Protein Gels, 1.0 mm, 9-well 10 gels NP0327BOX NuPAGE 4–12% Bis-Tris Protein Gels, 1.5 mm, 10-well 10 gels NP0335BOX NuPAGE 4–12% Bis-Tris Protein Gels, 1.5 mm, 10-well 2 gels NP0335PK2 NuPAGE 4–12% Bis-Tris Protein Gels, 1.5 mm, 15-well 10 gels NP0336BOX NuPAGE 4–12% Bis-Tris Protein Gels, 1.5 mm, 15-well 2 gels NP0336PK2 80 Product Quantity Cat. No. NuPAGE Bis-Tris midi gels (8 x 13 cm) NuPAGE 10% Bis-Tris Midi Protein Gels, 12+2 well 10 gels WG1201BOX NuPAGE 10% Bis-Tris Midi Protein Gels, 12+2-well, w/ adapters 10 gels WG1201A NuPAGE 10% Bis-Tris Midi Protein Gels, 20-well 10 gels WG1202BOX NuPAGE 10% Bis-Tris Midi Protein Gels, 20-well, w/ adapters 10 gels WG1202A NuPAGE 10% Bis-Tris Midi Protein Gels, 26-well 10 gels WG1203BOX NuPAGE 10% Bis-Tris Midi Protein Gels, 26-well, w/ adapters 10 gels WG1203A NuPAGE 4–12% Bis-Tris Midi Protein Gels, 12+2-well 10 gels WG1401BOX NuPAGE 4–12% Bis-Tris Midi Protein Gels, 12+2-well, w/ adapters 10 gels WG1401A NuPAGE 4–12% Bis-Tris Midi Protein Gels, 20-well 10 gels WG1402BOX NuPAGE 4–12% Bis-Tris Midi Protein Gels, 20-well, w/ adapters 10 gels WG1402A NuPAGE 4–12% Bis-Tris Midi Protein Gels, 26-well 10 gels WG1403BOX NuPAGE 4–12% Bis-Tris Midi Protein Gels, 26-well, w/ adapters 10 gels WG1403A NuPAGE 8% Bis-Tris Midi Protein Gels, 12+2-well 10 gels WG1001BOX NuPAGE 8% Bis-Tris Midi Protein Gels, 12+2-well, w/ adapters 10 gels WG1001A NuPAGE 8% Bis-Tris Midi Protein Gels, 20-well 10 gels WG1002BOX NuPAGE 8% Bis-Tris Midi Protein Gels, 20-well, w/ adapters 10 gels WG1002A NuPAGE 8% Bis-Tris Midi Protein Gels, 26-well 10 gels WG1003BOX NuPAGE 8% Bis-Tris Midi Protein Gels, 26-well, w/ adapters 10 gels WG1003A Product Quantity Cat. No. NuPAGE Bis-Tris midi gels (8 x 13 cm), WedgeWell format NuPAGE 10%, Bis-Tris Midi Protein Gels, 12+2-well, WedgeWell format 10 gels WBT01012BOX NuPAGE 10%, Bis-Tris Midi Protein Gels, 20-well, WedgeWell format 10 gels WBT01020BOX NuPAGE 10%, Bis-Tris Midi Protein Gels, 26-well, WedgeWell format 10 gels WBT01026BOX NuPAGE 4–12%, Bis-Tris Midi Protein Gels, 12+2-well, WedgeWell format 10 gels WBT41212BOX NuPAGE 4–12%, Bis-Tris Midi Protein Gels, 20-well, WedgeWell format 10 gels WBT41220BOX NuPAGE 4–12%, Bis-Tris Midi Protein Gels, 26-well, WedgeWell format 10 gels WBT41226BOX NuPAGE Tris-Acetate midi gels (8 x 13 cm), WedgeWell format NuPAGE 3–8% Tris-Acetate Midi Protein Gels, 12+2-well, WedgeWell format 10 gels WTA03812BOX NuPAGE 3–8% Tris-Acetate Midi Protein Gels, 20-well, WedgeWell format 10 gels WTA03820BOX NuPAGE 3–8% Tris-Acetate Midi Protein Gels, 26-well, WedgeWell format 10 gels WTA03826BOX Product Quantity Cat. No. Novex Tris-Glycine mini gels (8 x 8 cm), WedgeWell format Novex 6% Tris-Glycine Mini Gel, WedgeWell format, 10-well 10 gels XP00060BOX Novex 6% Tris-Glycine Mini Gel, WedgeWell format, 12-well 10 gels XP00062BOX Novex 6% Tris-Glycine Mini Gel, WedgeWell format, 15-well 10 gels XP00065BOX Novex 8% Tris-Glycine Mini Gel, WedgeWell format, 10-well 10 gels XP00080BOX Novex 8% Tris-Glycine Mini Gel, WedgeWell format, 12-well 10 gels XP00082BOX Novex 8% Tris-Glycine Mini Gel, WedgeWell format, 15-well 10 gels XP00085BOX Novex 10% Tris-Glycine Mini Gel, WedgeWell format, 10-well 10 gels XP00100BOX Novex 10% Tris-Glycine Mini Gel, WedgeWell format, 12-well 10 gels XP00102BOX Novex 10% Tris-Glycine Mini Gel, WedgeWell format, 15-well 10 gels XP00105BOX Novex 10% Tris-Glycine Mini Gel, WedgeWell format, 10-well 2 gels XP00100PK2 Novex 12% Tris-Glycine Mini Gel, WedgeWell format, 10-well 10 gels XP00120BOX Novex 12% Tris-Glycine Mini Gel, WedgeWell format, 12-well 10 gels XP00122BOX Novex 12% Tris-Glycine Mini Gel, WedgeWell format, 15-well 10 gels XP00125BOX Novex 14% Tris-Glycine Mini Gel, WedgeWell format, 10-well 10 gels XP00140BOX Novex 14% Tris-Glycine Mini Gel, WedgeWell format, 12-well 10 gels XP00142BOX Novex 14% Tris-Glycine Mini Gel, WedgeWell format, 15-well 10 gels XP00145BOX Novex 16% Tris-Glycine Mini Gel, WedgeWell format, 10-well 10 gels XP00160BOX Novex 16% Tris-Glycine Mini Gel, WedgeWell format, 12-well 10 gels XP00162BOX Novex 16% Tris-Glycine Mini Gel, WedgeWell format, 15-well 10 gels XP00165BOX Novex 4–12% Tris-Glycine Mini Gel, WedgeWell format, 10-well 10 gels XP04120BOX Novex 4–12% Tris-Glycine Mini Gel, WedgeWell format, 12-well 10 gels XP04122BOX Novex 4–12% Tris-Glycine Mini Gel, WedgeWell format, 15-well 10 gels XP04125BOX Novex 4–12% Tris-Glycine Mini Gel, WedgeWell format, 10-well 2 gels XP04120PK2 Novex 4–20% Tris-Glycine Mini Gel, WedgeWell format, 10-well 10 gels XP04200BOX Novex 4–20% Tris-Glycine Mini Gel, WedgeWell format, 12-well 10 gels XP04202BOX Novex 4–20% Tris-Glycine Mini Gel, WedgeWell format, 15-well 10 gels XP04205BOX Novex 4–20% Tris-Glycine Mini Gel, WedgeWell format, 10-well 2 gels XP04200PK2 Novex 8–16% Tris-Glycine Mini Gel, WedgeWell format, 10-well 10 gels XP08160BOX Novex 8–16% Tris-Glycine Mini Gel, WedgeWell format, 12-well 10 gels XP08162BOX Novex 8–16% Tris-Glycine Mini Gel, WedgeWell format, 15-well 10 gels XP08165BOX Novex 10–20% Tris-Glycine Mini Gel, WedgeWell format, 10-well 10 gels XP10200BOX Novex 10–20% Tris-Glycine Mini Gel, WedgeWell format, 12-well 10 gels XP10202BOX Novex 10–20% Tris-Glycine Mini Gel, WedgeWell format, 15-well 10 gels XP10205BOX 81 Product Quantity Cat. No. Novex Tris-Glycine Plus midi gels (8 x 13 cm) Novex 10% Tris-Glycine Plus Midi Protein Gels, 12+2-well 10 gels WPX01012BOX Novex 10% Tris-Glycine Plus Midi Protein Gels, 20-well 10 gels WXP01020BOX Novex 10% Tris-Glycine Plus Midi Protein Gels, 26-well 10 gels WXP01026BOX Novex 10% Tris-Glycine Plus Midi Protein Gels, 26-well, w/ adapters 10 gels WXP01026BOXA Novex 12% Tris-Glycine Plus Midi Protein Gels, 12+2-well 10 gels WXP01212BOX Novex 12% Tris-Glycine Plus Midi Protein Gels, 12+2-well, w/ adapters 10 gels WXP01212BOXA Novex 12% Tris-Glycine Plus Midi Protein Gels, 20-well 10 gels WXP01220BOX Novex 12% Tris-Glycine Plus Midi Protein Gels, 20-well, w/ adapters 10 gels WXP01220BOXA Novex 12% Tris-Glycine Plus Midi Protein Gels, 26-well 10 gels WXP01226BOX Novex 12% Tris-Glycine Plus Midi Protein Gels, 26-well, w/ adapters 10 gels WXP01226BOXA Novex 4–12% Tris-Glycine Plus Midi Protein Gels, 12+2-well 10 gels WXP41212BOX Novex 4–12% Tris-Glycine Plus Midi Protein Gels, 12+2-well, w/ adapters 10 gels WXP41212BOXA Novex 4–12% Tris-Glycine Plus Midi Protein Gels, 20-well 10 gels WXP41220BOX Novex 4–12% Tris-Glycine Plus Midi Protein Gels, 20-well, w/ adapters 10 gels WXP41220BOXA Novex 4–12% Tris-Glycine Plus Midi Protein Gels, 26-well 10 gels WXP41226BOX Novex 4–12% Tris-Glycine Plus Midi Protein Gels, 26-well, w/ adapters 10 gels WXP41226BOXA Novex 4–20% Tris-Glycine Plus Midi Protein Gels, 12+2-well 10 gels WXP42012BOX Novex 4–20% Tris-Glycine Plus Midi Protein Gels, 12+2-well, w/ adapters 10 gels WXP42012BOXA Novex 4–20% Tris-Glycine Plus Midi Protein Gels, 20-well 10 gels WXP42020BOX Novex 4–20% Tris-Glycine Plus Midi Protein Gels, 20-well, w/ adapters 10 gels WXP42020BOXA Novex 4–20% Tris-Glycine Plus Midi Protein Gels, 26-well 10 gels WXP42026BOX Novex 4–20% Tris-Glycine Plus Midi Protein Gels, 26-well, w/ adapters 10 gels WXP42026BOXA Novex 8–16% Tris-Glycine Plus Midi Protein Gels, 12+2-well 10 gels WXP81612BOX Novex 8–16% Tris-Glycine Plus Midi Protein Gels, 12+2-well, w/ adapters 10 gels WXP81612BOXA Novex 8–16% Tris-Glycine Plus Midi Protein Gels, 20-well 10 gels WXP81620BOX Novex 8–16% Tris-Glycine Plus Midi Protein Gels, 20-well, w/ adapters 10 gels WXP81620BOXA Novex 8–16% Tris-Glycine Plus Midi Protein Gels, 26-well 10 gels WXP81626BOX Novex 8–16% Tris-Glycine Plus Midi Protein Gels, 26-well, w/ adapters 10 gels WXP81626BOXA Product Quantity Cat. No. NativePAGE gels NativePAGE 3–12% Bis-Tris Protein Gels, 1.0 mm, 10-well 10 gels BN1001BOX NativePAGE 4–16% Bis-Tris Protein Gels, 1.0 mm, 10-well 10 gels BN1002BOX NativePAGE 3–12% Bis-Tris Protein Gels, 1.0 mm, 15-well 10 gels BN1003BOX NativePAGE 4–16% Bis-Tris Protein Gels, 1.0 mm, 15-well 10 gels BN1004BOX Novex Tricine gels Novex 10% Tricine Protein Gels, 1.0 mm, 10-well 10 gels EC6675BOX Novex 10% Tricine Protein Gels, 1.0 mm, 12-well 10 gels EC66752BOX Novex 16% Tricine Protein Gels, 1.0 mm, 10-well 10 gels EC6695BOX Novex 16% Tricine Protein Gels, 1.0 mm, 12-well 10 gels EC66952BOX Novex 16% Tricine Protein Gels, 1.0 mm, 15-well 10 gels EC66955BOX Novex 10–20% Tricine Protein Gels, 1.0 mm, 10-well 10 gels EC6625BOX Novex 10–20% Tricine Protein Gels, 1.0 mm, 12-well 10 gels EC66252BOX Novex 10–20% Tricine Protein Gels, 1.0 mm, 15-well 10 gels EC66255BOX Novex IEF gels Novex pH 3–7 IEF Protein Gels, 1.0 mm, 12-well 5 gels EC66452BOX Novex pH 3–7 IEF Protein Gels, 1.0 mm, 10-well 5 gels EC6645BOX Novex pH 3–10 IEF Protein Gels, 1.0 mm, 10-well 5 gels EC6655BOX Product Quantity Cat. No. Novex Tris-Glycine Plus midi gels (8 x 13 cm), WedgeWell format Novex 10% Tris-Glycine Plus Midi Protein Gels, 12+2-well, WedgeWell format 10 gels WTG01012BOX Novex 10% Tris-Glycine Plus Midi Protein Gels, 20-well, WedgeWell format 10 gels WTG01020BOX Novex 10% Tris-Glycine Plus Midi Protein Gels, 26-well, WedgeWell format 10 gels WTG01062BOX Novex 12% Tris-Glycine Plus Midi Protein Gels, 12+2-well, WedgeWell format 10 gels WTG01212BOX Novex 12% Tris-Glycine Plus Midi Protein Gels, 20-well, WedgeWell format 10 gels WTG01220BOX Novex 12% Tris-Glycine Plus Midi Protein Gels, 26-well, WedgeWell format 10 gels WTG01226BOX Novex 4–12% Tris-Glycine Plus Midi Protein Gels, 12+2-well, WedgeWell format 10 gels WTG41212BOX Novex 4–12% Tris-Glycine Plus Midi Protein Gels, 20-well, WedgeWell format 10 gels WTG41220BOX Novex 4–12% Tris-Glycine Plus Midi Protein Gels, 26-well, WedgeWell format 10 gels WTG41226BOX Novex 4–20% Tris-Glycine Plus Midi Protein Gels, 12+2-well, WedgeWell format 10 gels WTG42012BOX Novex 4–20% Tris-Glycine Plus Midi Protein Gels, 20-well, WedgeWell format 10 gels WTG42020BOX Novex 4–20% Tris-Glycine Plus Midi Protein Gels, 26-well, WedgeWell format 10 gels WTG42026BOX 82 Product Quantity Cat. No. Novex Zymogram Plus gels Novex 10% Zymogram Plus (Gelatin) Protein Gels, 1.0 mm, 15-well 10 gels ZY00105BOX Novex 10% Zymogram Plus (Gelatin) Protein Gels, 1.0 mm, 12-well 10 gels ZY00102BOX Novex 10% Zymogram Plus (Gelatin) Protein Gels, 1.0 mm, 10-well 10 gels ZY00100BOX E-PAGE High-Throughput Gel System E-PAGE 8% Protein Gels, 48-well 8 gels EP04808 E-Holder Platform 2 units EH03 E-PAGE Loading Buffer 1 4.5 mL EPBUF01 E-PAGE 6% Protein Gels, 96-well 8 gels EP09606 E-Gel Power Snap Electrophoresis System 1 unit G8300 Handcast polyacrylamide gels SureCast Gel Handcast Bundle A Multiple HC1000SR SureCast Gel Handcast Bundle B Multiple HC1000S SureCast Gel Handcast Station 1 casting station HC1000 SureCast Glass Plates 2 sets HC1001 SureCast Sealing Pads 2 pads HC1002 SureCast Multi-Use Tool, 10-well 1 unit HC1010 SureCast Multi-Use Tool, 12-well 1 unit HC1012 SureCast Multi-Use Tool, 15-well 1 unit HC1015 SureCast Gel Spacers 10 spacers HC1003 SureCast Stacking Buffer 2 x 500 mL dry packs HC2112 SureCast Stacking Buffer 5 x 500 mL dry packs HC2115 SureCast Resolving Buffer 2 x 500 mL dry packs HC2212 SureCast Resolving Buffer 5 x 500 mL dry packs HC2215 SureCast APS 25 g HC2005 SureCast Acrylamide Solution, 40% 450 mL HC2040 SureCast TEMED 30 mL HC2006 83 Product Quantity Cat. No. SDS-PAGE buffers Pierce SDS-PAGE Sample Prep Kit 50 reactions 89888 Bolt Transfer Buffer (20X) 125 mL BT0006 Bolt Transfer Buffer (20X) 1 L BT00061 4X Bolt LDS Sample Buffer 10 mL B0007 20X Bolt MES SDS Running Buffer 500 mL B0002 20X Bolt MES SDS Running Buffer 5 L B0002-02 20X Bolt MOPS SDS Running Buffer 500 mL B0001 20X Bolt MOPS SDS Running Buffer 5 L B0001-02 Bolt Antioxidant 15 mL BT0005 NuPAGE Tris-Acetate SDS Running Buffer (20X) 500 mL LA0041 NuPAGE MOPS SDS Running Buffer (20X) 500 mL NP0001 NuPAGE MOPS SDS Running Buffer (20X) 5 L NP000102 NuPAGE MOPS SDS Running Buffer (Powder) 5 packets NP000205 NuPAGE MES SDS Running Buffer (20X) 5 L NP000202 NuPAGE MES SDS Running Buffer (20X) 500 mL NP0002 NuPAGE MES SDS Running Buffer (Powder) 5 packets NP000105 Novex Tris-Glycine SDS Running Buffer (10X) 4 x 1 L LC26754 Novex Tris-Glycine SDS Running Buffer (10X) 500 mL LC2675 Novex Tris-Glycine SDS Running Buffer (10X) 5 L LC26755 Novex Rapid Tris-Glycine SDS Running Buffer (Powder) 5 packets LC2678 Novex Tricine SDS Running Buffer (10X) 500 mL LC1675 NuPAGE LDS Sample Buffer (4X) 10 mL NP0007 Novex Tricine SDS Sample Buffer (2X) 20 mL LC1676 Novex Tris-Glycine SDS Sample Buffer (2X) 20 mL LC2676 Novex Tris-Glycine Transfer Buffer (25X) 500 mL LC3675 NuPAGE Transfer Buffer (20X) 125 mL NP0006 NuPAGE Transfer Buffer (20X) 1 L NP00061 NuPAGE Antioxidant 15 mL NP0005 Novex Tris-Glycine SDS Buffer Kit 1 kit LC2677 NuPAGE MOPS SDS Buffer Kit (for Bis-Tris Gels) 1 kit NP0050 NuPAGE MES SDS Buffer Kit (for Bis-Tris Gels) 1 kit NP0060 NuPAGE Tris-Acetate SDS Buffer Kit (for Tris-Acetate Gels), contains 1 each LA0041, NP0004, NP0005, and NP0007 1 kit LA0050 Novex Tricine SDS Buffer Kit, includes LC1676 and LC1675 1 kit LC1677 Pierce LDS Sample Buffer, Nonreducing (4X) 5 mL 84788 Pierce Lane Marker Nonreducing Sample Buffer 5 mL 39001 Pierce 10X Tris-Glycine SDS Buffer 1 L 28362 BupH Tris-Glycine Buffer Packs 40 packs 28380 Product Quantity Cat. No. Native electrophoresis buffers Novex Tris-Glycine Native Running Buffer (10X) 500 mL LC2672 Novex Tris-Glycine Native Sample Buffer (2X) 20 mL LC2673 NativePAGE Running Buffer (20X) 1 L BN2001 NativePAGE Running Buffer Kit 1 kit BN2007 NativePAGE Cathode Buffer Additive (20X) 250 mL BN2002 NativePAGE Sample Buffer (4X) 10 mL BN2003 NativePAGE 5% G-250 Sample Additive 0.5 mL BN2004 NativePAGE Sample Prep Kit 1 kit BN2008 DDM (n-dodecyl β-D-maltoside) (10%) 1 mL BN2005 Digitonin (5%) 1 mL BN2006 Zymography buffers Novex Zymogram Developing Buffer (10X) 500 mL LC2671 Novex Zymogram Renaturing Buffer (10X) 500 mL LC2670 IEF buffers Novex IEF Anode Buffer (50X) 100 mL LC5300 Novex IEF Cathode Buffer pH 3–10 (10X) 125 mL LC5310 Novex IEF Cathode Buffer pH 3–7 (10X) 125 mL LC5370 Novex pH 3–10 IEF Buffer Kit, includes LC5300, LC5310, LC5311 1 kit LC5317 Novex pH 3–7 IEF Buffer Kit, includes LC5300, LC5370, LC5371 1 kit LC5377 Novex IEF Sample Buffer pH 3–10 (2X) 25 mL LC5311 Novex IEF Sample Buffer pH 3–7 (2X) 25 mL LC5371 84 Product Quantity Cat. No. Electrophoresis chamber systems and power supplies Mini Gel Tank 1 unit A25977 XCell SureLock Mini-Cell 1 unit EI0001 SureLock Tandem Midi Gel Tank 1 each STM1001 XCell4 SureLock Midi-Cell 1 each WR0100 PowerEase Touch 350W Power Supply (115 VAC) 1 each PS0350 PowerEase Touch 350W Power Supply (230 VAC) 1 each PS0351 PowerEase Touch 120W Power Supply (115 VAC) 1 each PS0120 PowerEase Touch 120W Power Supply (230 VAC) 1 each PS0121 Product Quantity Cat. No. Unstained standards HiMark Unstained Protein Standard 250 μL LC5688 PageRuler Unstained Low Range Protein Ladder 2 x 250 μL 26632 PageRuler Unstained High Range Protein Ladder 2 x 250 μL 26637 PageRuler Unstained Protein Ladder 2 x 250 μL 26614 NativeMark Unstained Protein Standard 5 x 50 μL LC0725 Prestained standards PageRuler Prestained Protein Ladder, 10–180 kDa 2 x 250 μL 26616 PageRuler Prestained Protein Ladder, 10–180 kDa 10 x 250 μL 26617 PageRuler Plus Prestained Protein Ladder, 10–250 kDa 2 x 250 μL 26619 PageRuler Plus Prestained Protein Ladder, 10–250 kDa 10 x 250 μL 26620 Spectra Multicolor Broad Range Protein Ladder 2 x 250 μL 26634 Spectra Multicolor Broad Range Protein Ladder 10 x 250 μL 26623 HiMark Prestained Protein Standard 250 μL LC5699 Spectra Multicolor Low Range Protein Ladder 250 μL 26628 Spectra Multicolor High Range Protein Ladder 2 x 250 μL 26625 SeeBlue Prestained Protein Standard 500 μL LC5625 SeeBlue Plus2 Prestained Protein Standard 500 μL LC5925 Western standards iBright Prestained Protein Ladder 2 x 250 mL LC5615 PageRuler Prestained NIR Protein Ladder 2 x 250 μL 26635 MagicMark XP Western Protein Standard 250 μL LC5602 MagicMark XP Western Protein Standard 50 μL LC5603 Specialty standards BenchMark Fluorescent Protein Standard 125 μL LC5928 BenchMark His-Tagged Protein Standard 125 μL LC5606 IEF Marker 3–10 500 μL 3921201 CandyCane Glycoprotein Molecular Weight Standards 400 μL C21852 PeppermintStick Phosphoprotein Molecular Weight Standards 400 μL P33350 Product Quantity Cat. No. Coomassie stains PageBlue Protein Staining Solution 1 L 24620 SimplyBlue SafeStain 1 L LC6060 SimplyBlue SafeStain 3.5 L LC6065 Imperial Protein Stain 1 L 24615 Imperial Protein Stain 3 x 1 L 24617 Silver stains Pierce Silver Stain Kit 1 L 24612 SilverXpress Silver Staining Kit 1 kit LC6100 Pierce Silver Stain for Mass Spectrometry 1 L 24600 Fluorescent and specialty stains SYPRO Orange Protein Gel Stain 500 μL S6650 SYPRO Orange Protein Gel Stain 10 x 50 μL S6651 SYPRO Red Protein Gel Stain 500 μL S6653 SYPRO Red Protein Gel Stain 10 x 50 μL S6654 SYPRO Ruby Protein Gel Stain 1 L S12000 SYPRO Ruby Protein Gel Stain 200 mL S12001 SYPRO Ruby Protein Gel Stain 5 L S21900 Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain 1 kit P21875 Pro-Q Diamond Phosphoprotein Gel Stain 1 L P33300 Pro-Q Diamond Phosphoprotein Gel Stain 200 mL P33301 Pro-Q Diamond Phosphoprotein Gel Stain 5 L P33302 No-Stain Protein Labeling Reagent 10 reactions A44717 No-Stain Protein Labeling Reagent 40 reactions A44449 References 1. Ornstein L (1964) Disc electrophoresis. 1. Background and theory. Ann N Y Acad Sci 121:321–349. 2. Moos M Jr, Nguye NY, Liu TY (1988) Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J Biol Chem 263:6005–6008. 3. Kubo K (1995) Effect of incubation of solutions of proteins containing dodecyl sulfate on the cleavage of peptide bonds by boiling. Anal Biochem 225:351–353. 85 Notes 86 97.4 kDa 97.4 kDa 97.4 kDa 97.4 kDa 97.4 kDa 97.4 kDa 97.4 kDa 97.4 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 200 kDa 200 kDa 200 kDa 200 kDa 200 kDa 200 kDa 200 kDa 200 kDa 260 kDa 116 kDa 116 kDa 116 kDa 116 kDa 116 kDa 116 kDa 116 kDa 116 kDa 160 kDa 66.3 kDa 66.3 kDa 66.3 kDa 66.3 kDa 66.3 kDa 66.3 kDa 66.3 kDa 66.3 kDa 55.4 kDa 55.4 kDa 55.4 kDa 55.4 kDa 55.4 kDa 55.4 kDa 55.4 kDa 55.4 kDa 36.5 kDa 36.5 kDa 36.5 kDa 36.5 kDa 36.5 kDa 36.5 kDa 36.5 kDa 36.5 kDa 31 kDa 31 kDa 31 kDa 31 kDa 31 kDa 31 kDa 31 kDa 31 kDa 14.4 kDa 14.4 kDa 14.4 kDa 14.4 kDa 14.4 kDa 14.4 kDa 14.4 kDa 6 kDa 6 kDa 6 kDa 6 kDa 6 kDa 2.5 kDa 2.5 kDa 3.5 kDa 2.5 kDa 3.5 kDa 3.5 kDa 3.5 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 3.5 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 3.5 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 3.5 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 80 kDa 260 kDa 160 kDa 21.5 kDa 21.5 kDa 21.5 kDa 21.5 kDa 21.5 kDa 21.5 kDa 21.5 kDa 10 20 30 40 50 60 70 80 90 %oflengthofgel 0 8% MES 8% MOPS 10% MES 10% MOPS 12% MES 12% MOPS 4–12% MES 4–12% MOPS 8% MES 8% MOPS 10% MES 10% MOPS 4–12% MES 4–12% MOPS 12% MES Denaturingseparation Denaturingseparation Invitrogen™ Bolt™ Bis-Tris Plus Gels Invitrogen™ NuPAGE™ Bis-Tris Gels Protein mini gel migration chart Use this chart to compare migration patterns of proteins of various molecular weights across the Invitrogen™ precast gel product line. 87 97 kDa 97 kDa 200 kDa 116 kDa 66 kDa 66 kDa 55 kDa 97 kDa 116 kDa 66 kDa 55 kDa 55 kDa 36 kDa 36 kDa 31 kDa 6 kDa 240 kDa 290 kDa 500 kDa 160 kDa 40 kDa 97 kDa 116 kDa 66 kDa 55 kDa 240 kDa 290 kDa 500 kDa 160 kDa 40 kDa 14 kDa 14 kDa 110 kDa 60 kDa 50 kDa 40 kDa 30 kDa 20 kDa 15 kDa 10 kDa 80 kDa 260 kDa 160 kDa 200 kDa 116 kDa 31 kDa 21 kDa 97 kDa 66 kDa 55 kDa 36 kDa 200 kDa 116 kDa 31 kDa 21 kDa 14 kDa 97 kDa 66 kDa 66 kDa 55 kDa 36 kDa 200 kDa 1,048 kDa 1,048 kDa 720 kDa 720 kDa 480 kDa 480 kDa 242 kDa 242 kDa 146 kDa 1,048 kDa 1,236 kDa 720 kDa 480 kDa 242 kDa 146 kDa 66 kDa 1,048 kDa 720 kDa 480 kDa 242 kDa 146 kDa 116 kDa 31 kDa 21 kDa 6 kDa 14 kDa 97 kDa 66 kDa 55 kDa 36 kDa 200 kDa 116 kDa 31 kDa 21 kDa 20 kDa 97 kDa 66 kDa 55 kDa 36 kDa 200 kDa 116 kDa 31 kDa 21 kDa 97 kDa 200 kDa 116 kDa 66 kDa 55 kDa 36 kDa 31 kDa 14 kDa 6 kDa 2.5 kDa 3.5 kDa 21 kDa 97 kDa 200 kDa 116 kDa 66 kDa 55 kDa 36 kDa 31 kDa 14 kDa 6 kDa 2.5 kDa 3.5 kDa 21 kDa 12% MOPS 10% 12% 4–12% 8–16% 4–20% 3–8% 7% 10% 16% 10–20% 3–8% 7% 3–12% 4–16% Denaturingseparation Denaturing separation Native separation Native separation Blotting and sequencing Synthetic peptides and tryptic analysis Wide range NuPAGE Tris-Acetate Gels Invitrogen™ NativePAGE™ Gels Invitrogen™ Novex™ Tris-Glycine Gels, WedgeWell™ format Invitrogen™ Novex™ Tricine Gels Invitrogen™ NuPAGE™ Tris-Acetate Gels For Research Use Only. 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